Factor xia-specific aptamers

ABSTRACT

The present disclosure concerns aptamers of formula (I) capable of specifically binding to Factor XIa. The aptamers can be used to prevent, treat or alleviate the symptoms of thrombosis. The aptamers can also be used to detect Factor XIa in a sample and/or purify Factor XIa from a sample. The aptamers can further be used to identity putative therapeutic agents for the prevention, treatment or alleviation of symptoms associated with thrombosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/331,626 filed May 4, 2016, the entire contents of which disclosure isspecifically incorporated herein by reference without disclaimer.

TECHNOLOGICAL FIELD

The present disclosure relates to compounds capable of specificallybinding to and limiting the biological activity of Factor XIa.

BACKGROUND

Thromboembolic diseases are a leading cause of morbidity and mortalityin the developed world and are rapidly emerging in the developing world.Currently commercialized antithrombotic agents are usually associatedwith the side effect of causing bleeding. In spite of the introductionof new oral anticoagulants into the clinic in the last few years (e.g.,dabigatran etexilate, rivaroxaban, edoxaban for example) there remainunmet needs.

Factor XI inhibition has been identified as an attractive target fordrug development because mice or humans with abnormally low FXI levelsappear to be protected from thrombosis. An antisense oligonucleotide toFXI named FXI-ASO (ISIS416858) is under clinical development (Buller etal., 2014) and was determined to be superior in preventing venousthromboembolism in patients undergoing total knee replacement than thestandard of care. Importantly, the study demonstrated that the antisenseoligonucleotide did not promote bleeding in orthopedic surgery, a majorchallenge with any antithrombic agent. However, it took five weeks forthe antisense oligonucleotide FXI-ASO (ISIS416858) to reduce FXI levelsby 10 to 20% of normal (e.g. pre-treatment) levels and FXI levels werealso slow to recover after the drug was stopped (Buller et al., 2014).

It would be highly desirable to be provided with a specific inhibitor ofFactor XIa (FXIa), e.g., a compound which would specifically bind to(and inhibit the biological activity of) Factor XIa. It would also bedesirable to be provided with a FXIa inhibitor, which would have a rapidonset of action and/or a short duration of action for treating and/orpreventing thrombosis. It would further be highly desirable to beprovided with a FXIa inhibitor which would exhibit little to noimmunogenicity (upon administration to the intended recipient).

BRIEF SUMMARY

The present disclosure concerns Factor XIa-specific aptamers. Theaptamers of the present disclosure are also capable of limiting thebiological activity (e.g., the proteolytic activity) of Factor XIa.

In a first aspect, the present disclosure provides an aptamer having thestructure of formula (I):

5′-5W—C-3W-3′  (I)

wherein:

-   -   “-” refers to a nucleotide bond;    -   5W has the following first nucleic acid sequence:

(SEQ ID NO: 66) N₁₇N₁₈N₁₉N₂₀N₂₁N₂₂N₂₃N₂₄N₇₁N₂₆N₂₇N₂₈AN₂₉TN₃₀TA;

-   -   C has the following second nucleic acid sequence:

(SEQ ID NO: 68) 5′-AACCTATN₁N₂N₃ACTATTN₄TN₅AN₆TN₇ATTTTTAN₈AN₉-3′;

-   -   3W can be present or absent and when present has the following        third nucleic acid sequence:

(SEQ ID NO: 67) 5′-N₃₁N₃₂N₃₃N₃₄N₃₅N₃₆N₃₇N₃₈N₃₉N₄₀N₄₁-3′;

-   -   the nucleotides at positions 14 to 18 of SEQ ID NO: 66 are        present;    -   the nucleotides at positions 1 to 33 of SEQ ID NO: 68 are        present;    -   the nucleotides at position 1 to 2 (AA) of SEQ ID NO: 68 are        capable of base pairing with nucleotides at position 28 to 27        (TT) of SEQ ID NO: 68;    -   the nucleotides at position 6 to 8 (ATC) of SEQ ID NO: 68 are        capable of base pairing with the nucleotides at position 25 to        23 (TAG) of SEQ ID NO: 68;    -   the nucleotides at position 11 to 14 (ACTA) of SEQ ID NO: 68 are        capable of base pairing with the nucleotides at position 22 to        19 (TN₆AN₅) of SEQ ID NO: 68; and    -   the nucleotides at position 14 to 18 (N₂₉TN₃₀TA) of SEQ ID NO:        66 are capable of base pairing with the nucleotides at position        33 to 29 (N₉AN₈AT) of SEQ ID NO: 68.

In such embodiment, the aptamer can have or consists essentially of thenucleic acid sequence of SEQ ID NO: 64. In still another embodiment, thenucleotides at positions 12 and 13 of SEQ ID NO: 66 and at position 34and 35 of SEQ ID NO: 68 are present, and the nucleotides at position 12and 13 (N₂₈A) of SEQ ID NO: 66 are capable of base pairing with thenucleotides at position 35 to 34 (N₁₀T) of SEQ ID NO: 68. In suchembodiment, the aptamer can have or consist essentially of the nucleicacid sequence of SEQ ID NO: 63. In still another embodiment, thenucleotides at position 8 to 11 of SEQ ID NO: 66, at position 1 to 5 ofSEQ ID NO: 67 and at position 36 of SEQ ID NO: 68 are present, and thenucleotide at position (N₂₄) of SEQ ID NO: 66 is capable of base pairingwith the nucleotide a position 2 (N₃₂) of SEQ ID NO: 67. In suchembodiment, the aptamer of can have or consist essentially of thenucleic acid sequence of SEQ ID NO: 62. In still another embodiment, thenucleotides at position 5 to 7 of SEQ ID NO: 66 and at position 3 to 5of SEQ ID NO: 67 are present, and the nucleotides at position 6 to 7(N₂₂N₂₃) of SEQ ID NO: 66 are capable of base pairing with thenucleotides at positions 4 to 3 (N₃₄N₃₃) of SEQ ID NO: 67. In suchembodiment, the aptamer can have or consist essentially of the nucleicacid sequence of SEQ ID NO: 61. In yet another embodiment, nucleotides 1to 4 of SEQ ID NO: 66 and nucleotides 6 to 11 of SEQ ID NO: 67 arepresent. In such embodiment, the aptamer can have or consist essentiallyof the nucleic acid sequence of SEQ ID NO: 60.

In an second aspect, the present disclosure provides an aptamer havingthe structure of formula (I) wherein:

-   -   5W has the following first nucleic acid sequence:

(SEQ ID NO: 25) 5′-N₁₂N₁₃N₁₄N₁₅N₁₆N₁₇N₁₈N₁₉N₂₀N₂₁N₂₂N₂₃N₂₄N₇₁N₂₆N₂₇N₂₈AN₂₉TN₃₀TA 3′;

-   -   C has the following second nucleotide sequence:

(SEQ ID NO: 23) 5′-AACCTATN₁N₂N₃ACTATTN₄TN₅AN₆TN₇ATTTTTAN₈AN₉TN₁₀N₁₁-3′.

-   -   3W is present and has the following third nucleotide sequence:

(SEQ ID NO: 27) 5′-N₃₁N₃₂N₃₃N₃₄N₃₅N₃₆N₃₇N₃₈N₃₉N₄₀N₄₁N₄₂N₄₃N₄₄N₄₅- 3′;

-   -   “-” refers to a nucleotide bond;    -   the nucleotides at position 1 to 36 of SEQ ID NO: 23, at        position 6 to 23 of SEQ ID NO: 25 and at position 1 to 11 of SEQ        ID NO: 27 are present;    -   the nucleotides at position 11 to 13 (ACTA) of SEQ ID NO: 23 are        capable of base pairing with the nucleotides at position 22 to        19 (TN₆AN₅) of SEQ ID NO: 23;    -   the nucleotides at position 6 to 8 (ATN₁) of SEQ ID NO: 23 are        capable of base pairing with the nucleotides at position 25 to        23 (TAN₇) of SEQ ID NO: 23;    -   the nucleotides at position 1 to 2 (AA) of SEQ ID NO: 23 are        capable of base pairing with the nucleotides at positions 28 to        27 (TT) of SEQ ID NO: 23;    -   the nucleotides at position 17 to 23 (N₂₈AN₂₉TN₃₀T) of SEQ ID        NO: 25 are capable of base pairing with the nucleotides at        positions 35 to 27 of SEQ ID NO: 23 (N₁₀TN₉AN₈A); and    -   the nucleotides at position 6 to 8 (N₂₂N₂₃N₂₄) of SEQ ID NO: 25        are capable of base pairing with the nucleotides at position 4        to 2 (N₃₄N₃₃N₃₂) of SEQ ID NO: 27.

In an embodiment, 5W has or consists essentially of the nucleotidesequence of SEQ ID NO: 26. In another embodiment, 3W has of consistsessentially of the nucleotide sequence of SEQ ID NO: 28. In stillanother embodiment, C has or consists essentially of the nucleotidesequence of SEQ ID NO: 24. In yet a further embodiment, the aptamer hasor consists essentially of the nucleotide sequence of SEQ ID NO: 1, SEQID SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11 or SEQ ID NO: 13.

In a third aspect, the present disclosure provides an aptamer having thenucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

In a fourth aspect, the present disclosure provides a variant of theaptamer described herewith.

In a fifth aspect, the present disclosure provides a fragment of theaptamer described herein.

In a sixth aspect, the present disclosure provides the aptamer describedherein, the variant described herein or the fragment described hereinfor use as a medicament, for example for preventing, treating oralleviating the symptoms of thrombosis. In an embodiment, the aptamer,variant or fragment are for the treatment of initial thrombosis. Inanother embodiment, the aptamer, variant or fragment are for theprevention of secondary thrombosis.

In a seventh aspect, the present disclosure provides, the aptamerdescribed herein, the variant described herein or the fragment describedherein for detecting Factor XIa in a biological sample.

In an eighth aspect, the present disclosure provides a method fordetecting Factor XIa in a sample. Broadly, the method comprises (i)contacting the aptamer described herein, the variant described herein orthe fragment described herein with the sample; (ii) determining thepresence or the absence of a complex between Factor XIa and the aptamer,the variant or the fragment; and (iii) detecting Factor XIa in thesample if the complex of step (ii) is determined to be present. In anembodiment, the method further comprises, when the complex of step (ii)is determined to be present, (iv) quantifying the amount of Factor XIain the sample based on the amount of the complex.

In a ninth aspect, the present disclosure comprises a method of imaginga clot in a subject. Broadly, the method comprises: (i) administeringthe aptamer described herein, the variant described herein or thefragment described herein to the subject; (ii) determining the presenceor the absence of a complex between Factor XIa and the aptamer, thevariant or the fragment; and (iii) imaging a clot in the subject at thelocation of the complex.

In a tenth aspect, the present disclosure comprises a method ofpurifying Factor XIa from a sample. Broadly, the method comprises: (i)contacting the aptamer described herein, the variant described herein orthe fragment described herein with the sample; (ii) allowing for acomplex between Factor XIa and the aptamer, the variant or the fragmentto form; and (iii) removing the complex from the sample.

In an eleventh aspect, the present disclosure comprises a method ofdetermining the usefulness of a test agent for the prevention, treatmentor the alleviation of symptoms of thrombosis in a subject. Broadly, themethod comprises: (i) contacting the test agent with Factor XIa toobtain a test level of the biological activity of Factor XIa, (ii)comparing the test level to a control level of the biological activityof Factor XIa, the control level being derived from or obtained bycontacting Factor XIa with the aptamer described herein, the variantdescribed herein or the fragment described herein; and (iii) determiningthe test agent as being useful for the prevention, treatment or thealleviation of symptoms of thrombosis if the test level is equal to orlower than the control level. In an embodiment, step (i) and/or (ii)further comprises determining the biological activity of Factor XIa bymeasuring the proteolytic activity of Factor XIa. In another embodiment,step (ii) further comprises providing the aptamer, the variant or thefragment and determining the control level of the biological activity ofFactor XIa.

In a twelfth aspect, the present disclosure comprises a method ofdetermining if a test aptamer is useful for the prevention, treatment orthe alleviations of symptoms of thrombosis in a subject. Broadly, themethod comprises: (i) contacting a test aptamer with Factor XIa toobtain a test level of the biological activity of Factor XIa, (ii)comparing the test level to a control level of the biological activityof Factor XIa, the control level being derived from or obtained bycontacting Factor XIa with the aptamer described herein, the variantdescribed herein or the fragment described herein; and (iii) determiningthe test aptamer as being useful for the prevention, treatment or thealleviation of symptoms of thrombosis if the test level is equal to orlower than the control level. In an embodiment, step (i) and/or (ii)further comprises determining the biological activity of Factor XIa bymeasuring the proteolytic activity of Factor XIa. In still anotherembodiment, step (ii) further comprises providing the aptamer, thevariant or the fragment and determining the control level of thebiological activity of Factor XIa. In still another embodiment, thecontrol level is derived from or obtained by contacting FXIa with acontrol aptamer having the nucleotide sequence of SEQ ID NO: 1. In yetanother embodiment, the test aptamer has at least one nucleotideaddition, substitution or deletion when compared to the control aptamerhaving the nucleotide sequence of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings, showing by way ofillustration, a preferred embodiment thereof, and in which:

FIGS. 1A and 1B illustrates how aptamers can be used as inhibitors ofFXIa-mediated amidolysis. (A) Means±SEM (n=3) of colour generationfollowing FXIa-mediated amidolysis of chromogenic substrate S2366, inthe presence (black bars) of Round 10 aptamers identified on the x axisor a scrambled negative control sequence (SCRAPT), or in the absence ofadded DNA molecules (white bar). (B) Mfold-generated predicted secondarystructure of FELIAP. The positions of base substitutions between FELIAPand other Round 10-selected aptamers are indicated by arrows.

FIGS. 2A to 2D provide the kinetic characterization of FELIAP inhibitionof FXIa-mediated amidolysis. (A) Means±SEM (n=3) of reaction velocityfor FXIa-mediated amidolysis of S2366 versus aptamer concentration(FELIAP, circles; SCRAPT, squares). (B) Means±SEM (n=3) of reactionvelocity versus S2366 concentration in the presence of 0, 10, or 20 μMFELIAP. (C) Lineweaver-Burke transformation of data in (B). (D)Means±SEM (n=7) of modified APTT assays in which FELIAP or SCRAPT werepre-incubated with FXIa, at concentrations given on the x axis, prior todilution into recalcified FXI-deficient plasma.

FIGS. 3A to 3C show the effects of FELIAP on reactions of FXIa and FXIwith macromolecular substrates. (A) Coomassie-stained reducedSDS-polyacrylamide gel. FIX was reacted with FXIa in the presence (+) orabsence (−) of SCRAPT (lane 2), FELIAP (lane 3) or KPI (lane 4). Arrowsand labels, at right, identify the position of FIX, forms of FIXa(including HC, heavy chain, HC+AP, heavy chain+activation peptideintermediate, LC, light chain) and KPI. M, markers (kDa), at left: 220;160; 120; 100; 90; 80; 70; 60; 50; 40; 30; 25; 20; 15. (B) As in (A),except antithrombin (AT, lane 1) was reacted with FXIa (lane 8) in thepresence of heparin for 1 (lane 2) or 5 (lane 3) minutes with FELIAP(lanes 4 and 5) or SCRAPT (lanes 6 and 7) or no DNA addition (lanes 2and 3). Arrows and labels identify the position of AT, FXIa-AT covalentcomplex, FXIa heavy chain (HC) and light chain (LC). M, markers (kDa),at left: 220; 160; 120; 100; 90; 80; 70; 60; 50; 40; 30; 25; 20 (C) Asin (A) and (B) except FXI and thrombin (IIa, lane 1) were reacted in theabsence (lane 2) or presence of dextran sulphate (DS, lanes 3-7) withthe addition of FELIAP (lane 4) or SCRAPT (lane 5) or the thrombininhibitor hirudin (lane 7). Lane 8, purified FXIa. M, markers (kDa), atleft: 220; 160; 120; 100; 90; 80; 70; 60; 50; 40; 30; 25; 20; 15.

FIGS. 4A to 41 show the inhibition of thrombin generation by FELIAP.Thrombin generation assays (TGA) were conducted in 3 different ways: inrecalcified human normal pooled plasma (NPP) using micronized silica(SIL) for contact activation as the initiator (NPP+SIL, panels A-C); inFXI-depleted plasma (FXI-DP) using tissue factor for extrinsic pathwayactivation as the initiator (FXI-DP+TF, panels D-F); and following 0.25nM FXIa preincubation with Buffer, 1 μM SCRAPT, or 1 μM FELIAP, inFXI-DP activated with micronized silica (FXI-DP+FXIa+SIL). Thrombinconcentration was determined fluorescently every minute for 60 minutesin each case. (A) TGA progress curves (mean±SD (n=6) with addition ofagents (Buffer, 30 μM FELIAP, or 30 μM SCRAPT) as indicated by labelsand arrows. Upwards error bars are shown. (B) As in (A), but n=5, andwith the addition of 2 μM recombinant hirudin variant 3 (hirudin). (C)As in (B), with the addition of agents (Buffer, 2 μM FELIAP, or 2 μMSCRAPT, or 2 μM KPI. Each set of thrombograms are quantified withrespect to endogenous thrombin potential (the area under the thrombogramcurve) (B, E, H) and time to peak thrombin (C, F, I) below the progresscurve panels. Bar graphs are derived from analysis of individualthrombogram curves corresponding to plasma supplementation with Buffer(white), SCRAPT (grey), FELIAP (black), or, in some reactions KPI orHirudin (light grey). Symbols (above the error bars) indicatestatistically significant differences from Buffer reactions, whilesymbols above the horizontal bar indicate statistically significantdifferences between SCRAPT- and FELIAP-supplemented and other reactions:p<0.001, **; p<0.001, ***.

FIG. 5 illustrates the binding of FELIAP to immobilized FXIa. An SPRsensogram showing the interactions between 3′biotinylated FELIAPimmobilized on a streptavidin-coated chip and FXIa at 25° C. at FXIaconcentrations given above each progress curves. Association was allowedto proceed for 180 seconds followed by 1800 seconds of dissociation. Allcurves were corrected for non-specific effects by signal subtractionusing a reference cell containing 3′ biotinylated SCRAPT immobilized ona streptavidin-coated chip. A single dilution series of curvesrepresentative of a total of 3 others is shown.

FIGS. 6A and 6B provide the characterization of truncated derivatives ofFELIAP as inhibitors of FXIa-mediated amidolysis. (A) Extent oftruncation analysis. Sequences to the left of bolded lines crossing theMfold-generated predicted secondary structure of FELIAP are identifiedas FELIAP_X, where X=the length of the truncated aptamer (32, 38, 42,49, 55, 64, or 74 for full-length FELIAP. (B) Means±SEM (n=3) of colourgeneration following FXIa-mediated amidolysis of chromogenic substrateS2366, in the presence (black bars) of SCRAPT, FELIAP, or truncatedderivatives of FELIAP identified in panels A and B, or in the absence ofof added DNA molecules (white bar).

DETAILED DESCRIPTION

In accordance with the present disclosure, there is provided aptamerscapable of specifically binding to FXIa. As the aptamers of the presentdisclosure were obtained using a negative selection with active-siteblocked FXIa, they are presumed to specifically bind to the active siteof FXIa and, ultimately limit FXIa's biological activity (e.g.,proteolytic activity). The aptamers of the present disclosure aredefined by their nucleotide sequence as well as their secondarystructure. Since FXIa is involved in the onset and maintenance ofthrombosis, the aptamers of the present disclosure can be used forpreventing, treating or alleviating the symptoms associated withthrombosis. Since the aptamers specifically bind to FXIa, they can alsobe used to determine the presence or the absence and, optionally theamount, of FXIa in a sample. Further, since the aptamers exhibitinhibitory activity towards FXIa, they can also be used as a control toscreen and identify therapeutic agents having improved antithromboticproperties.

Factor XIa-Specific Aptamers

The aptamers of the present disclosure specifically bind to (e.g., arespecific for) FXIa. In the context of the present disclosure, theexpressions “specific binding” or “specifically bind” refer to theinteraction between two elements in a manner that is determinative ofthe presence of the elements in the presence or absence of aheterogeneous population of molecules. For example, under designatedconditions, the aptamers of the present disclosure bind to FXIa and donot bind in a significant manner to other molecules (such as FXI orthrombin for example).

The aptamers of the present disclosure are single-stranded moleculescomposed of deoxyribonucleic acid (DNA) nucleotides, ribonucleic acid(RNA) nucleotides or a combination of both deoxyribonucleic andribonucleic acid nucleotides. In an embodiment, the aptamers of thepresent disclosure are exclusively made of deoxyribonucleic acid (DNA)nucleotides. The aptamers can be composed of naturally-occurringnucleobases (also referred to as bases), sugars and covalentinternucleoside (backbone) linkages. The aptamers can also have“non-naturally-occurring” or “synthetic” portions which functionsimilarly. In the context of the present disclosure, the term“nucleotides” refers to a deoxyribonucleic acid nucleotide or to aribonucleic acid nucleotides.

The aptamers of the present disclosure can include variousmodifications, e.g., stabilizing modifications, and thus can include atleast one modification in the phosphodiester linkage and/or on thesugar, and/or on the base. For example, the aptamer can include one ormore phosphorothioate linkages, phosphorodithioate linkages, and/ormethylphosphonate linkages. Different chemically compatible modifiedlinkages can be combined, e.g., modifications where the synthesisconditions are chemically compatible. While modified linkages areuseful, the aptamer can include phosphodiester linkages, e.g., includeat least one phosphodiester linkage, or at least 5%, 10%, 20%, 30% ormore phosphodiester linkages. Additional useful modifications include,without restriction, modifications at the 2′-position of the sugar (suchas 2′-O-alkyl modifications, 2′-O-methyl modifications, 2′-aminomodifications, 2′-halo modifications (e.g., 2′-fluoro) as well asacyclic nucleotide analogs. In another embodiment, the aptamer hasmodified linkages throughout, e.g., phosphorothioate; has a 3′- and/or5′-cap; includes a terminal 3′-5′ linkage.

In some embodiments, the aptamer includes a concatemer and comprises twoor more oligonucleotide sequences joined by one or more linker. Thelinker may, for example, consist of modified nucleotides ornon-nucleotide units. In some embodiments, the linker can provideflexibility to the aptamer. The use of concatemers as aptamers canprovide a facile method to synthesize a final molecule, by joiningsmaller oligonucleotides building blocks to obtain the desired length.For example, a 12 carbon linker (C₁₂ phosphoramidite) can be used tojoin two or more concatemers and provide length, stability andflexibility.

The aptamers of the present disclosure can include a natural or anon-natural backbone. Non-natural or synthetic backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates,carboranyl phosphate and borano-phosphates having normal 3′-5′ linkages,2′-5′ linked analogs of these, and those having inverted polaritywherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or2′ to 2′ linkage. Aptamers having inverted polarity typically include asingle 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. asingle inverted nucleoside residue which may be abasic (the nucleobaseis missing or has a hydroxyl group in place thereof). Some exemplarymodified aptamers' backbones that do not include a phosphodiesterlinkage have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts. Particularlyadvantageous are backbone linkages that include one or more chargedmoieties.

The aptamers of the present disclosure may also contain one or moresubstituted sugar moieties. For example, such oligonucleotides caninclude one of the following 2′-modifications: OH; F; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl, or2′-O—(O-carboran-1-yl)methyl. Particular examples areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)˜OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON [(CH₂)_(n)CH₃)]₂, where n and m arefrom 1 to 10. Other exemplary aptamers can include one of the following2′-modifications: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃,OCN, Cl, Br, CN, CF₃. OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl.

Other modifications to the aptamers include Locked Nucleic Acids (LNAs)in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom ofthe sugar ring thereby forming a bicyclic sugar moiety. Othermodifications include 2′-methoxy (2′-O—CH₃), 2′-methoxyethyl(2′O—CH₂—CH₃), 2′-ethyl, 2′-ethoxy, 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂),2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro(2′-F).

The 2′-modification may be in the arabino (up) position or ribo (down)position. Similar modifications may also be made at other positions onthe aptamers, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of the 5′ terminal nucleotide. The aptamers may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

The aptamers of the present disclosure can include “unmodified” or“natural” bases (nucleobases) such as adenine (A) and guanine (G), andthe pyrimidine bases thymine (T), cytosine (C) and uracil (U). Theaptamers may also include base modifications or substitutions. Modifiedbases include, but are not limited to other synthetic andnaturally-occurring bases such as 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—H₃) uraciland cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine. Modified bases may also include thosein which the purine or pyrimidine base is replaced with otherheterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine and 2-pyridone.

Another type of modification that can be included in the aptamers of thepresent disclosure are phosphorodithioate linkages. The aptamerscomprising modified oligonucleotides containing phosphorothioate ordithioate linkages may also contain one or more substituted sugarmoieties particularly modifications at the sugar moieties including,without restriction, 2′-ethyl, 2′-ethoxy, 2′-methoxy, 2′-aminopropoxy,2′-allyl, 2′-fluoro, 2′-pentyl, 2′-propyl, 2′-dimethylaminooxyethoxy,and 2′-dimethylaminoethoxyethoxy. The 2′-modification may be in thearabino (up) position or ribo (down) position. A preferred 2′-arabinomodification is 2′-fluoro. Similar modifications may also be made atother positions on the aptamer, particularly the 3′ position of thesugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotidesand the 5′ position of 5′ terminal nucleotide.

Even though the aptamers of the present disclosure are synthesized assingle-stranded molecules they can, under the appropriate conditions(e.g., salt, pH, temperature), form a secondary “hairpin” structure. Asshown in FIG. 1B, the aptamer FELIAP forms a hairpin structure in whichsome stretches of the molecule are capable of base-pairing (e.g.,capable of forming sections of double-stranded DNA and/or RNA) withother stretches of the molecule which are located several nucleotidesupstream or downstream. As also shown in FIG. 1B, the double-strandedconfiguration of FELIAP is not observed on the entire length of themolecule, but only in some regions. The aptamers of the presentdisclosure (which include FELIAP) are also capable of forming asecondary “hairpin” structure which is substantially similar to the oneshown on FIG. 1B. The aptamer of the present disclosure forms a hairpinstructure in which some stretches of the molecule are capable ofbase-pairing (e.g., capable of forming sections of double-stranded DNAand/or RNA) with other stretches of the molecule which may be locatedseveral nucleotides upstream or downstream. The double-strandedconfiguration of the aptamer of the present disclosure is not observedon the entire length of the molecule, but only in some regions.

The aptamers of the present disclosure can form at least one stretch(and in an embodiment a combination of stretches) of double stranded DNAand/or RNA between at the following positions:

-   -   the nucleotides at position 1 to 2 (AA) of SEQ ID NO: 68 with        the nucleotides at position 28 to 27 (TT) of SEQ ID NO: 68;    -   the nucleotides at position 6 to 8 (ATC) of SEQ ID NO: 68 with        the nucleotides at position 25 to 23 (TAG) of SEQ ID NO: 68;    -   the nucleotides at position 11 to 14 (ACTA) of SEQ ID NO: 68        with the nucleotides at position 22 to 19 (TN₆AN₅) of SEQ ID NO:        68;    -   the nucleotides at position 14 to 18 (N₂₉TN₃₀TA) of SEQ ID NO:        66 with the nucleotides at position 33 to 29 (N₉AN₈AT) of SEQ ID        NO: 68;    -   the nucleotides at position 12 and 13 (N₂₈A) of SEQ ID NO: 66        with the nucleotides at position 35 to 34 (N₁₀T) of SEQ ID NO:        68;    -   the nucleotide at position (N₂₄) of SEQ ID NO: 66 with the        nucleotide a position 2 (N₃₂) of SEQ ID NO: 67;    -   the nucleotides at position 6 to 7 (N₂₂N₂₃) of SEQ ID NO: 66        with the nucleotides at positions 4 to 3 (N₃₄N₃₃) of SEQ ID NO:        67;    -   the nucleotides at position 11 to 13 (ACTA) of SEQ ID NO: 23        with the nucleotides at position 22 to 19 (TN₆AN₅) of SEQ ID NO:        23;    -   the nucleotides at position 6 to 8 (ATN₁) of SEQ ID NO: 23 with        the nucleotides at position 25 to 23 (TAN₇) of SEQ ID NO: 23;    -   the nucleotides at position 1 to 2 (AA) of SEQ ID NO: 23 with        the nucleotides at positions 28 to 27 (TT) of SEQ ID NO: 23;    -   the nucleotides at position 17 to 23 (N₂₈AN₂₉TN₃₀T) of SEQ ID        NO: 25 with the nucleotides at positions 35 to 27 of SEQ ID NO:        23 (N₁₀TN₉AN₈A); and/or    -   the nucleotides at position 6 to 8 (N₂₂N₂₃N₂₄) of SEQ ID NO: 25        with the nucleotides at position 4 to 2 (N₃₄N₃₃N₃₂) of SEQ ID        NO: 27.

In the context of the present disclosure, the nucleotides which are notconsidered capable of base pairing with another nucleotide can form abulge structure and only form (covalent) bonds with adjacentnucleotides.

The aptamers of the present disclosure include more than contiguous 36nucleotides. In an embodiment, the aptamers of the present disclosureinclude at least 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77 or 78 contiguous nucleotides. Instill another embodiment, the aptamers of the present disclosurecomprise any range of nucleotides between 37 and 78 nucleotidescontiguous nucleotides. In yet a further embodiment, the aptamers of thepresent disclosure have 78 contiguous nucleotides.

The aptamers of the present disclosure have the structure of formula(I):

5W—C-3W  (I).

The structure of formula (I) comprises three main sections: a 5′ wing(referred to as “5W” in formula (I)), a core (referred to as “C” informula (I)) and an optional 3′ wing (referred to as “3W” in formula(I)). The 3′ end of the 5W section is associated (e.g., “-” which can bea covalent bond such as, for example, a nucleotide bond, a 5′ to 3′nucleotide bond for example) to the 5′ end of the C section of theaptamers of the present disclosure. The 3′ end of the C section isassociated (e.g., “-” which can be a covalent bond such as, for example,a nucleotide bond, a 5′ to 3′ nucleotide bond for example) to the 5′ endof the 3W section of the aptamers of the present disclosure. In someembodiments, the 3W section of the aptamers is absent.

The aptamers of the present disclosure are defined both by theirnucleotide sequence and their secondary structure. For example, some ofthe nucleotides of formula (I) are described of being capable of basepairing with another nucleotide. In the context of the presentdisclosure, a nucleotide is considered as “being capable of basepairing” with another nucleotide when they can form Watson-Crick basepairing. In such embodiment, C is considered of being capable of basepairing with G, G is considered of being capable of base pairing with C,A is considered of being capable of base pairing with T or U and T or Uare considered of being capable of base pairing with A. Othernucleotides of formula (I) are described as not being able to base pairwith another nucleotide. Still in the context of the present disclosure,a nucleotide is considered of “not being able to base pair” with anothernucleotide when they cannot form Watson-Crick base pairing. For example,C is not able of base pairing with C, A, T or U, G is not able of basepairing with G, A, T or U, A is not able of base pairing with A, C or G,T is not able of base pairing with C, G, T or U and U is not able ofbase pairing with C, G, T or U.

The core (C) section of the aptamers formula (I) has the followinggeneric nucleotide sequence (IIa) of (IIb):

(IIa - SEQ ID NO: 23) 5′-AACCTATN₁N₂N₃ACTATTN₄TN₅AN₆TN₇ATTTTTAN₈AN₉TN₁₀N₁₁-3′ or (IIb - SEQ ID NO: 68)5′-AACCTATN₁N₂N₃ACTATTN₄TN₅AN₆TN₇ATTTTTAN₈AN₉-3′

The core section of the aptamers of the present disclosure havenucleotides N₁ to N₁₁ which are each generic with respect to theidentity of the nucleotide base. The aptamers of the present disclosureinclude all combinations (e.g., generic and specific) of N₁ to N₁₁described herewith:

-   -   N₁ is located at position 8 of SEQ ID NO: 23 and 68 and is any        nucleotide (e.g., C, G, A, T/U), provided that it is capable of        base-pairing with N₇ (e.g., when N₁ is C, N₇ is G; when N₁ is G,        N₇ is C; when N₁ is A, N₇ is T or U; when N₁ is T or U, N₇ is        A). In an example, N₁ is V (e.g., not T/U). In yet another        example, N₁ can be A, C or G. In still another example, N₁ can        be C.    -   N₂ is located at position 9 of SEQ ID NO: 23 and 68 and is any        nucleotide (e.g., A, T/U, C or G). In an embodiment, N₂ can be K        (e.g., G or T/U). In still another embodiment, N₂ can be G.    -   N₃ is located at position 10 of SEQ ID NO: 23 and 68 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₃ can be K        (e.g., G or T/U). In another example, N₃ can be G.    -   N₄ is located at position 17 of SEQ ID NO: 23 and 68 and is V        (e.g., not T/U). For example, N₄ can be A, C or G. In yet        another example, N₄ can be G.    -   N₅ is located at position 19 of SEQ ID NO: 23 and 68 and is D        (e.g., not C). For example, N₅ can be A, G or T/U. In some        embodiments, N₅ can be T/U, such as, for example, T.    -   N₆ is located at position 21 of SEQ ID NO: 23 and 68 and is any        nucleotide (e.g., A, T/U, C or G). In an embodiment, N₆ can be K        (e.g., G or T/U). In yet another embodiment, N₆ can be G.    -   N₇ is located at position 23 of SEQ ID NO: 23 and 68 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₁ (e.g., when N₇ is C, N₁ is G; when N₇ is        G, N₁ is C; when N₇ is A, N₁ is T or U; when N₇ is T or U, N₁ is        A). In an embodiment, N₇ can be V (e.g., not T/U). In an        embodiment, N₇ can be A, C or G. For example, N₇ can be G.    -   N₈ is located at position 31 of SEQ ID NO: 23 and 68 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₃₀ of SEQ ID NO: 25 or 66 (e.g., when N₈        is C, N₃₀ is G; when N₈ is G, N₃₀ is C; when N₈ is A, N₃₀ is T        or U; when N₈ is T or U, N₃₀ is A). For example, N₈ can be K        (e.g., G or T/U). In yet another example, N₈ can be T/U, such        as, for example, T.    -   N₉ is located at position 33 of SEQ ID NO: 23 and 68 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₂₉ of SEQ ID NO: 25 or 66 (e.g., when N₉        is C, N₂₉ is G; when N₉ is G, N₂₉ is C; when N₉ is A, N₂₉ is T        or U; when N₉ is T or U, N₂₉ is A). In an embodiment, N₉ can be        K (e.g., G or T/U). In still another embodiment, N₉ can be G.    -   N₁₀ is located at position 35 of SEQ ID NO: 23 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₂₈ of SEQ ID NO: 25 or 66 (e.g., when N₁₀        is C, N₂₈ is G; when N₁₀ is G, N₂₈ is C; when N₁₀ is A, N₂₈ is T        or U; when N₁₀ is T or U, N₂₈ is A). For example, N₁₀ can be K        (e.g., G or T/U). In still another example, N₁₀ can be G.    -   N₁₁ is located at position 36 of SEQ ID NO: 23. N₁₁ can be        present or absent. When present, N₁₁ can be any nucleotide        (e.g., A, T/U, C or G). In an embodiment, N_(u) can be present.        In still yet another example, N₁₁ can be T.

A comparison of the core sections of some of the aptamers of the Exampleis presented in Table 1. In an embodiment, the core region of theaptamers of the present disclosure comprises or consists essentially ofany one of the nucleotide sequence of SEQ ID NO: 24, 30 to 35 and 69. Inyet another embodiment, the core region of the aptamers of the presentdisclosure comprises or consists essentially of any one of thenucleotide sequence of SEQ ID NO: 24, 30, 32, 35, 36 and 69. In stillanother embodiment, the core region of the aptamers of the presentdisclosure can include or consist essentially of the nucleotide sequenceof SEQ ID NO: 24.

The 5′ wing (5W) section of the aptamers of formula (I) has thefollowing generic nucleotide sequence of Formula (IIIa) or (IIIb):

(IIIa - SEQ ID NO: 25) 5′-N₁₂N₁₃N₁₄N₁₅N₁₆N₁₇N₁₈N₁₉N₂₀N₂₁N₂₂N₂₃N₂₄N₂₅N₂₆N₂₇N₂₈AN₂₉TN₃₀TA-3′ or (IIIb - SEQ ID NO: 66)5′-N₁₇N₁₈N₁₉N₂₀N₂₁N₂₂N₂₃N₂₄N₂₅N₂₆N₂₇N₂₈AN₂₉TN₃₀ TA-3′

The 5W section of the aptamers of the present disclosure havenucleotides which are each generic with respect to the identity of thenucleotide base. The aptamers of the present disclosure include allcombinations (e.g., generic and specific) of the nucleotides describedherewith.

In an embodiment, the 5W region can have or consist essentially of thenucleic acid sequence of SEQ ID: 25. The aptamers of the presentdisclosure include all combinations (e.g., generic and specific) of N₁₂to N₃₀ described herewith. In embodiments in which the 5W region has orconsists essentially of SEQ ID NO: 25, the following embodiments arecontemplated:

-   -   N₁₂ is located at position 1 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). In an example, N₁₂ can be G.    -   N₁₃ is located at position 2 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). In an example, N₁₃ can be A.    -   N₁₄ is located at position 3 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₁₄ can be A.    -   N₁₅ is located at position 4 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). In an embodiment, N₁₅ can be        T.    -   N₁₆ is located at position 5 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). In an example, N₁₆ can be T.    -   N₁₇ is located at position 6 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₁₇ can be C.    -   N₁₈ is located at position 7 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). In an embodiment, N₁₈ can be        T.    -   N₁₉ is located at position 8 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₁₉ can be A.    -   N₂₀ is located at position 9 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). In an embodiment, N₂₀ can be        A.    -   N₂₁ is located at position 10 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₂₁ can be T.    -   N₂₂ is located at position 11 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₃₄ of SEQ ID NO: 27 (e.g., when N₂₂ is C,        N₃₄ is G; when N₂₂ is G, N₃₄ is C; when N₂₂ is A, N₃₄ is T or U;        when N₂₂ is T or U, N₃₄ is A). In an embodiment, N₂₂ can be A.    -   N₂₃ is located at position 12 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₃₃ of SEQ ID NO: 27 (e.g., when N₂₃ is C,        N₃₃ is G; when N₂₃ is G, N₃₃ is C; when N₂₃ is A, N₃₃ is T or U;        when N₂₃ is T or U, N₃₃ is A). For example, N₂₃ can be C.    -   N₂₄ is located at position 13 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₃₂ of SEQ ID NO: 27 (e.g., when N₂₄ is C,        N₃₂ is G; when N₂₄ is G, N₃₂ is C; when N₂₄ is A, N₃₂ is T or U;        when N₂₄ is T or U, N₃₂ is A). For example, N₂₄ can be G.    -   N₂₅ is located at position 14 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₂₅ can be A.    -   N₂₆ is located at position 15 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₂₆ can be C.    -   N₂₇ is located at position 16 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G). For example N₂₇ can be T.    -   N₂₈ is located at position 17 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₁₀ of SEQ ID NO: 23 (e.g., when N₂₈ is C,        N₁₀ is G; when N₂₈ is G, N₁₀ is C; when N₂₈ is A, N₁₀ is T or U;        when N₂₈ is T or U, N₁₀ is A). For example, N₂₈ can be C.    -   N₂₉ is located at position 19 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₉ of SEQ ID NO: 23 (e.g., when N₂₉ is C,        N₉ is G; when N₂₉ is G, N₉ is C; when N₂₉ is A, N₉ is T or U;        when N₂₉ is T or U, N₉ is A). For example, N₂₉ can be C.    -   N₃₀ is located at position 21 of SEQ ID NO: 25 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₈ of SEQ ID NO: 23 (e.g., when N₃₀ is C,        N₈ is G; when N₃₀ is G, N₈ is C; when N₃₀ is A, N₈ is T or U;        when N₃₀ is T or U, N₈ is A). For example, N₃₀ can be A.

In another embodiment, the 5W region of the aptamers of the presentdisclosure include or consist essentially of of the nucleotide sequenceof GAATTCTAATACGACTCACTATA (SEQ ID NO: 26).

In an embodiment, the 5W region can be truncated (as provided in thenucleic acid sequence of SEQ ID NO: 66) and each nucleotides can begeneric with respect to the identity of the nucleotide base. Theaptamers of the present disclosure include all combinations (e.g.,generic and specific) of N₁₇ to N₃₀ described herewith. In embodimentsin which the 5W region has or consists essentially of SEQ ID NO: 66, thefollowing embodiments are contemplated:

-   -   N₁₇ is located at position 1 of SEQ ID NO: 66. It can be present        of absent. When present, it can be any nucleotide (e.g., A, T/U,        C or G). For example, N₁₇ can be C.    -   N₁₈ is located at position 2 of SEQ ID NO: 66. It can be absent        when N₁₇ is also absent. When present, it can be any nucleotide        (e.g., A, T/U, C or G). In an embodiment, N₁₈ can be T.    -   N₁₉ is located at position 3 of SEQ ID NO: 66. It can be absent        when N₁₇ and N₁₈ are also absent. It can be any nucleotide        (e.g., A, T/U, C or G). For example, N₁₉ can be A.    -   N₂₀ is located at position 4 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈ and N₁₉ are also absent. It can be any nucleotide        (e.g., A, T/U, C or G). In an embodiment, N₂₀ can be A.    -   N₂₁ is located at position 5 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉ and N₂₀ are also absent. It can be any        nucleotide (e.g., A, T/U, C or G). For example, N₂₁ can be T.    -   N₂₂ is located at position 6 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉, N₂₀ and N₂₁ are also absent. It can be any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₃₄ of SEQ ID NO: 67 (e.g., when N₂₂ is C,        N₃₄ is G; when N₂₂ is G, N₃₄ is C; when N₂₂ is A, N₃₄ is T or U;        when N₂₂ is T or U, N₃₄ is A). In an embodiment, N₂₂ can be A.    -   N₂₃ is located at position 7 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉, N₂₀, N₂₁ and N₂₂ are also absent. It can be        and is any nucleotide (e.g., A, T/U, C or G), provided that it        is capable of base pairing with N₃₃ of SEQ ID NO: 67 (e.g., when        N₂₃ is C, N₃₃ is G; when N₂₃ is G, N₃₃ is C; when N₂₃ is A, N₃₃        is T or U; when N₂₃ is T or U, N₃₃ is A). For example, N₂₃ can        be C.    -   N₂₄ is located at position 8 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉, N₂₀, N₂₁, N₂₂ and N₂₃ are also absent. It        can be and is any nucleotide nucleotide (e.g., A, T/U, C or G),        provided that it is capable of base pairing with N₃₂ of SEQ ID        NO: 67 (e.g., when N₂₄ is C, N₃₂ is G; when N₂₄ is G, N₃₂ is C;        when N₂₄ is A, N₃₂ is T or U; when N₂₄ is T or U, N₃₂ is A). For        example, N₂₄ can be G.    -   N₂₅ is located at position 9 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉, N₂₀, N₂₁, N₂₂, N₂₃ and N₂₄ are also absent.        It can be and is any nucleotide (e.g., A, T/U, C or G). For        example, N₇₁ can be A.    -   N₂₆ is located at position 10 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉, N₂₀, N₂₁, N₂₂, N₂₃, N₂₄ and N₂₅ are also        absent. It can be any nucleotide (e.g., A, T/U, C or G). For        example, N₂₆ can be C.    -   N₂₇ is located at position 11 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉, N₂₀, N₂₁, N₂₂, N₂₃, N₂₄, N₂₅ and N₂₆ are        also absent. It can be any nucleotide (e.g., A, T/U, C or G).        For example N₂₇ can be T.    -   N₂₈ is located at position 12 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉, N₂₀, N₂₁, N₂₂, N₂₃, N₂₄, N₂₅, N₂₆ and N₂₇        are also absent. It can be any nucleotide (e.g., A, T/U, C or        G), provided that it is capable of base pairing with N₁₀ of SEQ        ID NO: 67 (e.g., when N₂₈ is C, N₁₀ is G; when N₂₈ is G, N₁₀ is        C; when N₂₈ is A, N₁₀ is T or U; when N₂₈ is T or U, N₁₀ is A).        For example, N₂₈ can be C.    -   N₂₉ is located at position 14 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉, N₂₀, N₂₁, N₂₂, N₂₃, N₂₄, N₂₅, N₂₆, N₂₇ and        N₂₈ are also absent. It can be any nucleotide (e.g., A, T/U, C        or G), provided that it is capable of base pairing with N₉ of        SEQ ID NO: 67 (e.g., when N₂₉ is C, N₉ is G; when N₂₉ is G, N₉        is C; when N₂₉ is A, N₉ is T or U; when N₂₉ is T or U, N₉ is A).        For example, N₂₉ can be C.    -   N₃₀ is located at position 16 of SEQ ID NO: 66. It can be absent        when N₁₇, N₁₈, N₁₉, N₂₀, N₂₁, N₂₂, N₂₃, N₂₄, N₂₅, N₂₆, N₂₇, N₂₈        and N₂₉ are also absent. It can be any nucleotide (e.g., A, T/U,        C or G), provided that it is capable of base pairing with N₈ of        SEQ ID NO: 67 (e.g., when N₃₀ is C, N₈ is G; when N₃₀ is G, N₈        is C; when N₃₀ is A, N₈ is T or U; when N₃₀ is T or U, N₈ is A).        For example, N₃₀ can be A.

In embodiments in which the 5W region has the nucleic acid sequence ofSEQ ID NO: 66, it is provided that the 5W region can correspond toresidues 14 to 18, 12 to 18, 5 to 18 or 1 to 18.

The “3′ wing” (3W) section of the aptamers of formula (I) has thefollowing generic nucleotide sequence of Formula (IVa) or (IVb):

(IVa - SEQ ID NO: 27) 5′-N₃₁N₃₂N₃₃N₃₄N₃₅N₃₆N₃₇N₃₈N₃₉N₄₀N₄₁N₄₂N₄₃N₄₄N₄₅-3′ or (IVb - SEQ ID NO: 67) 5′-N₃₁N₃₂N₃₃N₃₄N₃₅N₃₆N₃₇N₃₈N₃₉N₄₀N₄₁-3′

The 3W section of the aptamers of the present disclosure havenucleotides which are each generic with respect to the identity of thenucleotide base. The aptamers of the present disclosure include allcombinations (e.g., generic and specific) of the nucleotides describedherewith. In embodiments in which the the 3W region has or consistsessentially of SEQ ID NO: 27, the following embodiments arecontemplated:

-   -   N₃₁ is located at position 1 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₃₁ can be G.    -   N₃₂ is located at position 2 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₂₄ of SEQ ID NO: 25 (e.g., when N₃₂ is C,        N₂₄ is G; when N₃₂ is G, N₂₄ is C; when N₃₂ is A, N₂₄ is T or U;        when N₃₂ is T or U, N₂₄ is A). For example, N₃₂ can be C.    -   N₃₃ is located at position 3 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G), provided that it is capable        of base pairing with N₂₃ of SEQ ID NO: 25 (e.g., when N₃₃ is C,        N₂₃ is G; when N₃₃ is G, N₂₃ is C; when N₃₃ is A, N₂₃ is T or U;        when N₃₃ is T or U, N₂₃ is A). For example, N₃₃ can be G.    -   N₃₄ is located at position 4 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G), provided that it can base        pair with N₂₂ of SEQ ID NO: 25 (e.g., when N₃₄ is C, N₂₂ is G;        when N₃₄ is G, N₂₂ is C; when N₃₄ is A, N₂₂ is T or U; when N₃₄        is T or U, N₂₂ is A). For example, N₃₄ can be T.    -   N₃₅ is located at position 5 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₃₅ can be C.    -   N₃₆ is located at position 6 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₃₆ can be C.    -   N₃₇ is located at position 7 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₃₇ can be A.    -   N₃₈ is located at position 8 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₃₈ can be A.    -   N₃₉ is located at position 9 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₃₉ can be C.    -   N₄₀ is located at position 10 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₄₀ can be A.    -   N₄₁ is located at position 11 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₄₁ can be C.    -   N₄₂ is located at position 12 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₄₂ can be A.    -   N₄₃ is located at position 13 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₄₃ can be T.    -   N₄₄ is located at position 14 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₄₄ can be C.    -   N₄₅ is located at position 15 of SEQ ID NO: 27 and is any        nucleotide (e.g., A, T/U, C or G). For example, N₄₅ can be G.

In an embodiment, the 3W region of the aptamers of the presentdisclosure include or consist essentially of the nucleotide sequence ofGCGTCCAACACATCG (SEQ ID NO: 28).

In an embodiment, the 3W region can be truncated (as provided in thenucleic acid sequence of SEQ ID NO: 67) and each nucleotides can begeneric with respect to the identity of the nucleotide base. Theaptamers of the present disclosure include all combinations (e.g.,generic and specific) of N₃₁ to N₄₁ described herewith. In embodimentsin which the 5W region has or consists essentially of SEQ ID NO: 67, thefollowing embodiments are contemplated:

-   -   N₃₁ is located at position 1 of SEQ ID NO: 67. It can be absent        if N₃₂, N₃₃, N₃₄, N₃₅, N₃₆, N₃₇, N₃₈, N₃₉, N₄₀ and N₄₁ are        absent. It can be and is any nucleotide (e.g., A, T/U, C or G).        For example, N₃₁ can be G.    -   N₃₂ is located at position 2 of SEQ ID NO: 67. It can be absent        if N₃₃, N₃₄, N₃₅, N₃₆, N₃₇, N₃₈, N₃₉, N₄₀ and N₄₁ are absent. It        can be any nucleotide (e.g., A, T/U, C or G), provided that it        is capable of base pairing with N₂₄ of SEQ ID NO: 66 (e.g., when        N₃₂ is C, N₂₄ is G; when N₃₂ is G, N₂₄ is C; when N₃₂ is A, N₂₄        is T or U; when N₃₂ is T or U, N₂₄ is A). For example, N₃₂ can        be C.    -   N₃₃ is located at position 3 of SEQ ID NO: 67. It can be absent        if N₃₄, N₃₅, N₃₆, N₃₇, N₃₈, N₃₉, N₄₀ and N₄₁ are absent. It can        be any nucleotide (e.g., A, T/U, C or G), provided that it is        capable of base pairing with N₂₃ of SEQ ID NO: 66 (e.g., when        N₃₃ is C, N₂₃ is G; when N₃₃ is G, N₂₃ is C; when N₃₃ is A, N₂₃        is T or U; when N₃₃ is T or U, N₂₃ is A). For example, N₃₃ can        be G.    -   N₃₄ is located at position 4 of SEQ ID NO: 67. It can be absent        if N₃₅, N₃₆, N₃₇, N₃₈, N₃₉, N₄₀ and N₄₁ are absent. It can be        any nucleotide (e.g., A, T/U, C or G), provided that it can base        pair with N₂₂ of SEQ ID NO: 66 (e.g., when N₃₄ is C, N₂₂ is G;        when N₃₄ is G, N₂₂ is C; when N₃₄ is A, N₂₂ is T or U; when N₃₄        is T or U, N₂₂ is A). For example, N₃₄ can be T.    -   N₃₅ is located at position 5 of SEQ ID NO: 67. It can be absent        if N₃₆, N₃₇, N₃₈, N₃₉, N₄₀ and N₄₁ are absent. It can be any        nucleotide (e.g., A, T/U, C or G). For example, N₃₅ can be C.    -   N₃₆ is located at position 6 of SEQ ID NO: 67. It can be absent        if N₃₇, N₃₈, N₃₉, N₄₀ and N₄₁ are absent. It can be any        nucleotide (e.g., A, T/U, C or G). For example, N₃₆ can be C.    -   N₃₇ is located at position 7 of SEQ ID NO: 67. It can be absent        if N₃₈, N₃₉, N₄₀ and N₄₁ are absent. It can be any nucleotide        (e.g., A, T/U, C or G). For example, N₃₇ can be A.    -   N₃₈ is located at position 8 of SEQ ID NO: 67. It can be absent        if N₃₉, N₄₀ and N₄₁ are absent. It can be any nucleotide (e.g.,        A, T/U, C or G). For example, N₃₈ can be A.    -   N₃₉ is located at position 9 of SEQ ID NO: 67. It can be absent        if N₄₀ and N₄₁ are absent. It can be any nucleotide (e.g., A,        T/U, C or G). For example, N₃₉ can be C.    -   N₄₀ is located at position 10 of SEQ ID NO: 67. It can be absent        if N₄₁ is absent. It can be any nucleotide (e.g., A, T/U, C or        G). For example, N₄₀ can be A.    -   N₄₁ is located at position 11 of SEQ ID NO: 67. It can be        absent. It can be any nucleotide (e.g., A, T/U, C or G). For        example, N₄₁ can be C.

In embodiments in which the 3W region has the nucleic acid sequence ofSEQ ID NO: 67, it is provided that the 3W region can correspond toresidues 1 and 2, 1 to 5 or 1 to 11.

The aptamers of the present disclosure forms a modified “hairpin”structure comprising both regions of double stranded (e.g., Watson-Crickbase paired) nucleotides (e.g., stems) and regions of single strandednucleotides (e.g., bulges). The “stem” regions refer to regions of theaptamers that are capable of base pairing and, under the appropriateconditions, form double stranded regions. Since the aptamers are in anhairpin configuration, the stem regions involve base pairing of a firstsection in the 5′→3′ orientation with a second section in the 3′→5′orientation. The “bulge” regions refer to regions of the aptamers whichremain single stranded and, due to the secondary structure of the restof the aptamers, protrude outward from the stem regions. The aptamers ofthe present disclosure have at least one stem region and at least onebulge region. In an embodiment, the aptamers of the present disclosurecomprise four “stem” regions and five “bulge” regions. In suchembodiments, each stem region is flanked (on each side) by a bulgeregion.

The aptamers of formula (I) can have up to four stem regions (designatedherein as a first, a second, a third and a fourth stem region) and thenucleotides which are not located in the stem regions (e.g., theremaining nucleotides) are considered to be located in one of the bulgeregions. As such, the nucleotides at position 3 to 5, 9, 10, 15 to 18,26 and 36 of SEQ ID NO: 23 (as well as the corresponding positions inSEQ ID NO: 68) lack the ability to base pair (e.g., cannot base pair)with any other nucleotides of the aptamers Formula (I) and are in asingle stranded configuration. In addition, the nucleotides at position1 to 10 and 14 to 16 of SEQ ID NO: 25 (as well the correspondingpositions in SEQ ID NO: 66) lack the ability to base pair (e.g., cannotbase pair) with any other nucleotides of the aptamers Formula (I) andare in a single stranded configuration. Further, the nucleotides atposition 1 and 5 to 15 of SEQ ID NO: 27 (as well as the correspondingpositions in SEQ ID NO: 67) lack the ability to base pair with any othernucleotides of Formula (I) and are in a single stranded configuration.

The first stem region of the aptamers of Formula (I) involves the basepairing of the nucleotides located at position 11 to 14 of SEQ ID NO: 23or 68 with the nucleotides located at positions 22 to 19 of SEQ ID NO:23 or 68. As such, in the aptamers of formula (I), the nucleotides atposition 11 to 14 of SEQ ID NO: 23 or 68 are capable of base pairingwith nucleotides at position 22 to 19 of SEQ ID NO: 23 or 68.

The first stem region can be flanked by a first bulge region and asecond bulge region. The first bulge region can consist of nucleotideslocated at positions 15 to 18 of SEQ ID NO: 23 or 68. The second bulgeregion can consist of nucleotides located at positions 9 and 10 of SEQID NO: 23 or 68.

The second stem region of the aptamers of Formula (I) involves the basepairing of the nucleotides located at position 6 to 8 of SEQ ID NO: 23or 68 with the nucleotides located at positions 17 to 23 of SEQ ID NO:23 or 68. As such, in the aptamers of formula (I), the nucleotides atposition 6 to 8 of SEQ ID NO: 23 or 68 are capable of base pairing withthe nucleotides at position 17 to 23 of SEQ ID NO: 23 or 68.

The second stem region can be flanked by the second bulge region(described above) and a third bulge region. The third bulge region canconsist of nucleotides at positions 2 to 4 and 26 of SEQ ID NO: 23 or68.

The third stem region of the aptamers of formula (I) involves the basepairing of the nucleotides located at position 17 to 23 of SEQ ID NO: 25(or position 12 to 18 of SEQ ID NO: 66) and at positions 1 and 2 of SEQID NO: 23 or 68 with the nucleotides located at positions 35 to 27 ofSEQ ID NO: 23 or 68. As such, in the aptamers of formula (I), thenucleotides at position 19 to 23 of SEQ ID NO: 25 (or position 12 to 18of SEQ ID NO: 66) and at position 1 and 2 of SEQ ID NO: 23 or 68 arecapable of base pairing with the nucleotides at positions 35 to 27 ofSEQ ID NO: 23 or 68.

The third stem region can flanked by the third bulge region (asindicated above) and a fourth bulge region. The fourth bulge region canconsist of the nucleotides at position of 14 to 16 SEQ ID NO: 25 (orposition 9 to 11 of SEQ ID NO: 66), the nucleotide at position 36 of SEQID NO: 23 or 68 and the nucleotide at position 1 of SEQ ID NO: 27 or 67.

The fourth stem region of the aptamers of formula (I) involves the basepairing of the nucleotides located at position 11 to 13 of SEQ ID NO: 25(or position 6 to 8 of SEQ ID NO: 66) with the nucleotides located atpositions 4 to 2 of SEQ ID NO: 27 or 67. As such, in the aptamers offormula (I), the nucleotides at position 11 to 13 of SEQ ID NO: 25 (orat position 6 to 8 of SEQ ID NO: 66) are capable of base pairing withthe nucleotides at position 4 to 2 of SEQ ID NO: 27 or 67.

The fourth stem regions can be flanked by the fourth bulge region (asindicated above) and a fifth bulge region. The fifth bulge region canconsist of the nucleotides at position 1 to 10 of SEQ ID NO: 25 (orposition 1 to 6 of SEQ ID NO: 66) and the nucleotides at position 15 to5 of SEQ ID NO: 27 (or position 11 to 5 of SEQ ID NO: 68).

The present disclosure also provides variants of the aptamers of Formula(I) provided that such variants are capable of specifically binding toFXIa. In some embodiments, the variants of the aptamers of Formula (I)can exhibit some FXIa inhibitory activity. A “variant” of the aptamersof Formula (I) (also referred to as an aptamer variant) has at least onenucleotide addition or substitution when compared to the aptamers ofFormula (I). The one or more added or substituted nucleotides that canbe located anywhere in the molecule. The level of identity between thevariants and the aptamers of Formula (I) is at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% over the entire length of the aptamers.In an embodiment, the variant aptamer has one or more of the stem andbuldge region as described herein.

The present disclosure further provides fragments of the aptamers ofFormula (I) and variants of the aptamers of Formula (I) provided thatsuch fragments retain the ability to specifically bind to FXIa. In someembodiments, the fragments of the aptamers of Formula (I) and of theaptamers of Formula (I) can exhibit some FXIa inhibitory activity. A“fragment” of the aptamers of Formula (I) (also referred to as anaptamer fragment) has at least one less nucleotide than the aptamers ofFormula (I) or the variants described herein. The one or morenucleotides that can be removed from the aptamers of Formula (I) toprovide the “fragments” can be located anywhere in the molecule. Forexample, the one or more nucleotide that can be removed from theaptamers of Formula (I) can be located, at the 5′ end of the molecule,at the vicinity of the 5′ end of the molecule, at the 3′ end of themolecule and/or at the vicinity of the 3′ end of the molecule. In anembodiment, the fragment is a 5′- and/or a 3′-end truncation of one ormore nucleotides. In some embodiments, the fragments of the aptamers ofFormula (I) have at least 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or 77 contiguous nucleotidesof the aptamers of Formula (I). In an embodiment, the aptamer fragmenthas one or more of the stem and buldge region as described herein.

Therapeutic Uses of the Factor XIa-Specific Aptamers

The FXIa-specific aptamers are capable of limiting (and in someembodiments, inhibiting) the activation of Factor IX (e.g., theproteolytic cleavage of Factor IX into Factor IXa). The FXIa-specificaptamers are capable of limiting (and in some embodiments, inhibiting)the generation of thrombin in plasma. Since the FXIa-specific aptamershave a rapid onset of action, they can be used in an acute setting toachieve therapeutic effects. In addition, since the FXIa-specificaptamers have a relatively short duration of therapeutic action (whencompared to FXI-specific antisense), they provide ease in ending thetherapy and limiting unwanted side effects. In some embodiment, theFXIa-specific aptamers of the present disclosure have a short durationof action, e.g. after administration, they exhibit their therapeuticactivity during one or more hours, during one or more days, but duringless than a week. Consequently, the FXIa-specific aptamers can be usedto prevent, treat or alleviate the symptoms of thrombosis (or athrombotic condition) in a subject in need thereof.

The Factor XIa-specific aptamers disclosed herein can be formulated intoa pharmaceutical composition for administration to a subject in needthereof. In an embodiment, the subject is a mammal, such as a human.More specifically, the FXIa-specific aptamers can be admixed with acarrier and formulated in a pharmaceutical composition. As used herein,a carrier or “pharmaceutically acceptable carrier” is a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more compounds to the subject, andis typically liquid or solid. A pharmaceutical carrier is generallyselected to provide the desired property (bulk, consistency, etc.), whencombined with components of a given pharmaceutical composition, in viewof the intended administration mode.

The FXIa-specific aptamers or the pharmaceutical composition comprisingsame may be administered in a unit dosage form. Conventionalpharmaceutical practice may be employed to provide suitable formulationsor compositions to administer such compositions to subjects. Althoughintravenous administration is preferred, any appropriate route ofadministration may be employed, for example, oral, parenteral,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intrathecal, epidural,intracisternal, intraperitoneal, intranasal, or aerosol administration.The FXIa aptamers or the pharmaceutical composition may be in the formof a liquid solution or a suspension for intravenous administration, inthe form of a tablet or a capsule for oral administration, in the formof a powder, nasal drops, or an aerosol for intranasal formulations.

In order to provide a therapeutic benefit, the FXIa-specific aptamers orthe pharmaceutical composition comprising same is administered at a“pharmaceutically/therapeutically effective amount”. The expressions“pharmaceutically effective amount” or “therapeutically effectiveamount” refers to an amount (dose) effective in preventing a conditionin a subject, treating a subject and/or alleviating its symptoms. It isalso to be understood herein that a “pharmaceutically effective amount”of the FXIa-specific aptamers can be interpreted as an amount giving adesired therapeutic effect, either taken in one dose or in any dosage orroute, taken alone or in combination with other therapeutic agents.

When used in therapy and a very specific window of exposure to theFXIa-specific aptamers is warranted (for example to limit the appearanceor maintenance of unwanted side effects), it is possible to use amodified FXIa-specific aptamer bearing a biotin group. The biotin groupcan be located at any position on the aptamer, but is preferably locatedat or near the 5′ or the 3′ end of the molecule. The biotin group itselfis not intended to reduce or dampen the therapeutic activity of theFXIa-specific aptamer but provides means for removing (inactivating) theaptamer of the present disclosure. In fact, in the presence of avidin,the biotin group is capable of forming a complex which will prevent theFXIa-specific aptamer from mediating its therapeutic activity (e.g.,limiting the biological activity of FXIa) by removing it from generalcirculation. As such, when therapeutic effects of the FXIa-specificaptamers are warranted, no avidin is administered to the treatedsubject. However, when the therapeutic effects of the FXIa-specificaptamers are no longer warranted, avidin can be administered to thetreated subject to reduce the therapeutic activity of the aptamers.

The FXIa-specific aptamers can be used to prevent, treat or alleviationthe symptoms of thrombosis (e.g. including initial thrombosis and asecondary thrombosis). The FXIa-specific aptamers can be used asthrombolytic agents, adjunct therapy or as anticoagulants. In someembodiments, the FXIa-specific aptamers can be used to treat an initialthrombosis (e.g., a first diagnosed thrombosis in a subject) and/orprevent a secondary thrombosis (e.g., a subsequent thrombosis in asubject).

In another embodiment, the FXIa-specific aptamers can be used asthrombolytic agents (e.g., indirect thrombolytic agents) for dissolvinga clot in vivo. In order to do so, a therapeutic dose of theFXIa-specific aptamers is administered to a subject in need thereof(having one or more clots and would benefit from reducing the sizeand/or number of the one or more clots). In some embodiments, theFXIa-specific aptamers can be used to treat thrombosis (or athrombotic-associated condition) in a subject in need thereof. Forexample, the FXIa-specific aptamers can be used to treat an initialthrombosis (e.g., a first thrombotic event in a subject). In suchembodiments, the FXIa-specific aptamers can be used alone, or incombination with another thrombolytic agent.

In another embodiment, the administration of the FXIa-specific aptamersor the pharmaceutical composition comprising same can be used as anadjunct therapy to bolster the effect of a thrombolytic agent. Suchagents include, but are not limited to, tPA, a tissue plasminogenactivator variant (such as, for example, tenecteplase), urokinase andstreptokinase. When used in combination with the FXIa-specific aptamers,it is possible to administer a lower dose of a thrombolytic agent, evena dose to be considered sub-therapeutic (when administered in theabsence of the FXIa-specific aptamers). In an additional or optionalembodiment, when used in combination with a FXIa-specific aptamer, itmay be possible to administer the thrombolytic agent at a time which isconsidered outside the effective window after the onset of symptoms(e.g. more than three hours after myocardial infarction or 5 hours afterstroke) and still observe beneficial therapeutic effect in the subject.

In yet another embodiment, the FXIa-specific aptamers are able to reducethe formation of a clot and as such can be used as anticoagulants. Inorder to do so, a therapeutic dose of the the FXIa-specific aptamers ofthe pharmaceutical composition comprising same is administered to asubject in need thereof (having a clot(s) and could benefit fromreducing the formation of the clot(s)). The FXIa-specific aptamers canbe used alone or in combination with another anticoagulant, such as, forexample, heparin or its derivatives. For example, the FXIa-specificaptamers can be used to prevent an initial thrombosis (e.g., a firstthrombotic event in a subject) in a subject at risk of developing theinitial thrombosis (e.g. a subject intended to be surgically operated).In another example, the FXIa-specific aptamers can be used to prevent asecondary thrombosis (e.g., a subsequent thrombotic event in thesubject).

Factor XIa-Specific Aptamer Methods

Since the aptamers of the present disclosure are able to specificallybind to Factor XIa, they can be used to either detect the presence ofFactor XIa or purify Factor XIa from a mixture comprising othercomponents. In the methods using the aptamers of the present disclosure,a label (either covalently associated or non-covalently associated) ispreferably used. In methods of detecting FXIa, the label is preferably adetectable label. In methods of purifying FXIa, the label is an affinitylabel.

In an embodiment, the FXIa-specific aptamers are used in a method todetect the presence and optionally quantify the amount of FXIa orlocalize FXIa. For example, the FXIa-specific aptamers can be used todetect the presence of FXIa in a sample. In the context of the presentdisclosure, a sample is mixture (either already in a liquid or capableof being provided as in a liquid form) suspected of comprising FXIa. Thesample can be a solution or a suspension. The sample can be processedinto a solution or a suspension. The sample can be a biological sample.Exemplary biological samples include, but are not limited to, bodilyfluids (e.g., blood, urine, gastro-intestinal juice, interstitial fluid,lachrymal fluid, sweat, saliva, stools, sputum, pus, cerebrospinalfluid, semen, prostatic fluid, milk, nipple aspirate fluid, lachrymalfluid, perspiration), tissues (swabs (e.g., cheek swabs), tissuebiopsy), fractionated bodily fluids (serum, plasma, etc.), cell extracts(e.g., cytoplasmic membrane, mitochondrial extract, nuclear extracts,etc.), cell suspensions, secretions as well as cultures of suchbiological samples. In order to detect the presence of FXIa in a sample,the aptamers of the present disclosure are admixed with the sample underconditions favoring the formation of a complex between the aptamers andFXIa. In such conditions, the presence of complex between the aptamerand FXIa is indicative of the presence of FXIa in the sample. Still insuch conditions, the absence of a complex between the aptamer and FXIais indicative of the absence of FXIa in the sample. In such method, itis possible to quantify the amount of the FXIa in the sample, especiallywhen the aptamer is modified to be associated with a detectable label(e.g., a radioactive label, an enzymatic label or a fluorescent labelfor example). By measuring the signal associated with the label, it ispossible to determine or estimate the amount complexes formed betweenthe aptamer and FXIa. Alternatively, the label is a solid support andthe solid support is washed from the unbound elements of the sample todetect and optionally quantify FXIa.

In another embodiment, the FXIa-specific aptamers can be used in animaging method to detect the presence and localize FXIa in a subject. Insuch embodiment, the aptamers of the present disclosure (preferablymodified to be associated with a detectable label such as a radioactivelabel) are administered to the subject under conditions to allow theformation of a complex between the aptamers and FXIa. Since FXIa ismostly located in a clot or in the vicinity of a clot, then, the subjectis then submitted to an imaging technique to determine if the detectablelabel associated with the aptamers localize in one or more areas in thesubject. The detection of the label in the individual is indicative ofthe presence (and optionally the localization) of one or more clots inthe subject.

In yet another embodiment, the FXIa-specific aptamers can be used toenrich or purify FXIa from a sample comprising other components thanFXIa. In such embodiments, the FXIa-specific aptamers are modifiedeither to bear or be associated with an affinity label. TheFXIa-specific aptamers are admixed with the sample (which is preferablya liquid sample) under conditions allowing for the formation of acomplex between the FXIa-specific aptamers and FXIa. Then, the complexis retrieved from the sample using the affinity label present on orassociated with the aptamers in order to enrich the concentration oreven purify FXIa from the sample. Alternatively, the label is a solidsupport and the solid support is washed from the unbound elements of thesample to enrich or purify FXIa.

Screening Assays Based on Factor XIa-Specific Aptamer

The present disclosure provides FXIa-specific aptamers which can be usedas controls to develop additional therapeutics, including additionalaptamers, having improved therapeutic or safety properties for theprevention, treatment or the alleviation of symptoms associated withthrombosis in a subject (such as a mammal, e.g., a human).

In an embodiment, the present disclosure provides a method ofdetermining if a putative therapeutic agent (herein referred to as atest agent) would be useful for the prevention, treatment or thealleviation of symptoms of thrombosis in a subject. In order to do so,the test agent is contacted with Factor XIa to obtain a test level ofthe biological activity of Factor XIa. The contacting step between thetest agent and FXIa can be done in vivo (e.g., in a non-human animal) orin vitro. Since FXIa's biological activity is a proteolytic activity,the test level can be obtained by measuring the proteolytic activity ofFXIa (in the presence of the test agent) be either determining theamount of an uncleaved substrate of FXIa or a proteolytic productgenerated by FXIa. In an embodiment, the substrate of FXIa is FIX andthe proteolytic product is FIXa. In another embodiment, the substrate isa synthetic substrate of FXIa (e.g., S2366) and the proteolytic productis a chromogenic or fluorescent label of the synthetic substrate. Oncethe test level has been determined, it is compared to a control level ofthe biological activity of FXIa. The control level can be derived fromor obtained by contacting FXIa with the aptamer described herein, theaptamer variant described herein or the aptamer fragment describedherein. In some embodiments, the FELIAP aptamer is used to obtain orderive the control level. Optionally, the method can comprisedetermining the control level of the biological activity of FXIa andproviding the aptamer, the aptamer variant or the aptamer fragment. Oncethe comparison is made, then it can be determined if the test agent isuseful for preventing, treating or alleviating the symptoms associatedwith thrombosis. If the test level is equal to or lower than the controllevel (e.g., if the test agent inhibits more the biological activity ofFXIa than the aptamers, the aptamer variants or the aptamer fragments),then it is determined that the test agent is useful for preventing,treating or alleviating the symptoms associated with thrombosis.However, if the test level is higher than the control level (e.g., ifthe test agent inhibits less the biological activity of FXIa than theaptamers, the aptamer variants or the aptamer fragments), then it isdetermined that the test agent is not useful for preventing, treating oralleviating the symptoms associated with thrombosis.

In another embodiment, the present disclosure provides a method of usingthe aptamers, the aptamer variants or the aptamer fragments describedherein as “leads” to screen for aptamers having improved properties. Assuch, the present disclosure also provides a method of determining if anaptamer (herein referred to as a test aptamer) would be useful for theprevention, treatment or the alleviation of symptoms of thrombosis in asubject. In some embodiments, the method can be used to screen a libraryof aptamers having at least one nucleotide substitution, addition ordeletion when compared to the aptamers/variants/fragments of the presentdisclosure. In order to do so, the test aptamer is contacted with FactorXIa to obtain a test level of the biological activity of Factor XIa. Thecontacting step between the test aptamer and FXIa can be done in vivo(e.g., in a non-human animal) or in vitro. Since FXIa's biologicalactivity is a proteolytic activity, the test level can be obtained bymeasuring the proteolytic activity of FXIa (in the presence of the testaptamer) be either determining the amount of an uncleaved substrate ofFXIa or a proteolytic product generated by FXIa. In an embodiment, thesubstrate of FXIa is FIX and the proteolytic product is FIXa. In anotherembodiment, the substrate is a synthetic substrate of FXIa (e.g., S2366)and the proteolytic product is a chromogenic or fluorescent label of thesynthetic substrate. Once the test level has been determined it iscompared to a control level of the biological activity of FXIa. Thecontrol level can be derived from or obtained by contacting FXIa withthe aptamer described herein, the aptamer variant described herein orthe aptamer fragment described herein. In some embodiments, the FELIAPaptamer is used to obtain or derive the control level. Optionally, themethod can comprise determining the control level of the biologicalactivity of FXIa and providing the aptamer, the aptamer variant or theaptamer fragment. Once the comparison is made, then it can be determinedif the test aptamer is useful for preventing, treating or alleviatingthe symptoms associated with thrombosis. If the test level is equal toor lower the control level (e.g., if the test aptamer inhibits more thebiological activity of FXIa than the aptamers, the aptamer variants orthe aptamer fragments), then it is determined that the test aptamer isuseful for preventing, treating or alleviating the symptoms associatedwith thrombosis. However, if the test level is higher than the controllevel (e.g., if the test aptamer inhibits less the biological activityof FXIa than the aptamers, the aptamer variants or the aptamerfragments), then it is determined that the test aptamer is not usefulfor preventing, treating or alleviating the symptoms associated withthrombosis.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example

Reagents. The aptamer library comprised a ssDNA template with sequence5′-GAATTCTAAT ACGACTCACT ATA-N₄₀-GCGTCCAACA CATCG-3′ (SEQ ID NO: 29).The forward (A) and reverse primers (B) were 5′-GAATTCTAAT ACGACTCACTATA-3′ (SEQ ID NO: 40) and 5′-GCGTCCAACAC ATCG-3′ (SEQ ID NO: 41)respectively. These and all other oligonucleotides employed (see Table 2below for a description of their nucleic acid sequence) were purchasedfrom Integrated DNA Technologies (IDT, Coralville, Iowa). FXI, FXIa, FIXand FXIIa were bought from Enzyme Research Laboratories (South Bend,Ind.). Biotinylated goat anti-human Factor XI (FXI) antibody waspurchased from Affinity Biologicals (Ancaster, ON). Dynabeads BiotinBinder was bought from Thermo Fisher Scientific (Waltham, Mass.). Forthrombin generation assays (TGA), TGA substrate and TGA calibrator setswere obtained from Technothrombin GmbH (Vienna, Austria). ActivatedPartial Thromboplastin Time (APTT) reagent was from Diagnostica Stago(Asnieres, France). Chromogenic substrate S2366 was purchased fromInstrumentation Laboratory (Lexington, Mass.).

TABLE 2 Nucleotidic sequence of the tested aptamers. The underlinedregions of the sequences refer to the “constant primer binding” sites(e.g., the 5′ wing or the 3′ wing). The italicized regions represent the“variable” domains (e.g., the core region). FXIa inhibitory activity wasassessed as the inhibition of amidolytic activity of chromogenicsubstrate SEQ ID NOs are provided for the entire aptamer as well as thevaribale (core) region. S2366. +++ = highest FXIa inhibitory activity,++ = active FXIa inhibitory activity, + = slightly active FXIainhibitory activity, − = no FXIa inhibitory activity. FXIa inhib- itoryDesig- activ- SEQ nation Nucleotide sequence tiy ID NOs FELIAPGAATTCTAATACGACTCACTATA AACCTATCGGACTATTGTTAGTGATTTTTATAGTGTGCGTCCAACACATCG +++  1 and 24 NRMAPT 1 GAATTCTAATACGACTCACTATATACGTGGTTCTTTTTTTAGGGAGTTCGATCCTGAGGCCT GCGTCCAACACATCG +  2 APT10_EGAATTCTAATACGACTCACTATA TGTCACTCTGATCAAAAATTTTGTAGTCATCTTGTTATGCGCGTCCAACACATCG +  3 and 69 NRMAPT 3 GAATTCTAATACGACTCACTATACATAAAAACTATATACGTGGTTCTTTTTTTAGTTTTTCGT GCGTCCAACACATCG −  4 NRMAPT 4GAATTCTAATACGACTCACTATA TCTTACATGGCCCCATTATTTTAGAGTTCATTCCGATTGGGCGTCAAACACATCG −  5 APT10_D GAATTCTAATACGACTCACTATAAACCTATCGGACTATTGTTAGTGATTTTTAGAGT GGCGTCCAACACATCG +  6 and 30 NRMAPT 6GAATTCTAATACGACTCACTATA GCGTATACGTGGTTCTTTTTTCGCAGGATAGTATGTATTTGCGTCCAACACATCG −  7 NRMAPT 7 GAATTCTAATACGACTCACTATAAACCTATCGTACTATTGTTAGTGATTTTTATAGTGT GCGTCCAACACATCG −  8 and 31 APT10_AGAATTCTAATACGACTCACTATA AACCTATCGGACTATTGTTAGTGATTTTTATAGTTTGCGTCCAACACATCG ++  9 and 32 NRMAPT 9 GAATTCTAATACGACTCACTATAAACCTATCTGACTATTGTTAGTGATTTTTATAGTGT GCGTCCAACACATCG − 10 and 33NRMAPT10 GAATTCTAATACGACTCACTATA AACCTATCGGACTATTGTTATTGATTTTTATAGTGTGCGTCCAACACATCG − 11 and 34 NRMAPT11 GAATTCTAATACGACTCACTATACATAAAAACTATATACGTGGTTCTTTTTTTAGTTTTTCTT GCGTCCAACACATCG − 12 APT10_BGAATTCTAATACGACTCACTATA AACCTATCGGACTATTGTTAGTGATTTTTATATTGTGCGTCCAACACATCG ++ 13 and 35 APT10_C GAATTCTAATACGACTCACTATAAACCTATCGGACTATTTTTAGTGATTTTTATAGTGT GCGTCCAACACATCG − 14 and 36NRMAPT14 GAATTCTAATACGACTCACTATA AACCTATTGGACTATTGTTAGTGATTTTTATAGTGTGCGTCCAACACATCG − 15 and 37 NRMAPT15 GAATTCTAATACGACTCACTATACACGTGGTTCTTTATTTAGTTATGTCGTCGTTTTTTCAT GCGTCCAACACATCG − 16 NRMAPT16GAATTCTAATACGACTCACTATA AACCTATCGGACTATTGTTAGTTATTTTTATAGTGTGCGTCCAACACATCG − 17 and 38 NRMAPT17 GAATTCTAATACGACTCACTATAAACCTATCGGACTATTGTCAGTGATTTTTATAGTGT GCGTCCAACACATCG − 18 and 39NRMAPT18 GAATTCTAATACGACTCACTATACATAAAAACTATATACTTGGTTCTTTTTTTAGTTTTTCGT GCGTCCAACACATCG − 19 NRMAPT19GAATTCTAATACGACTCACTATA CACAAAAACTATATACCTGGTTCTTTTTTTAGTTTTTCGTGCGTCCAACACATCG − 20 NRMAPT20 GAATTCTAATACGACTCACTATAAACACACAAACCTATTTTTCGATTTTCCTGCCATCACTCC GCGTCCAACACATCG − 21 NRMAPT21GAATTCTAATACGACTCACTATA CATAAAAACTATATACGTTGTTCTTTTTTTAGTTTTTCGTGCGTCCAACACATCG − 22

Systemic Evolution of Ligands by Exponential Enrichment (SELEX).Solution-based SELEX was performed as previously described (Tuerk etal., 1990) with some modifications. In the sequence of the startingssDNA library, 5′-GAATTCTAAT ACGACTCACT ATA-N₄₀-GCGTCCAACA CATCG-3′ (SEQID NO: 29), N₄₀ represents a 40 nucleotide randomized region. Beforeselection, streptavidin-coated magnetic beads (5 μL packed volume) wereprewashed five times in Aptamer Folding Buffer (AFB; 20 mM Tris-HCl pH7.4, 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂ and 1 mM CaCl₂). All reactionstook place at room temperature; washes were performed using a magnet toconcentrate the beads at the bottom of the reaction tube. Next,biotinylated goat anti-human factor XI antibody (Affinity Biologicals)was added at quadruple the FXIa concentration to be employed, andincubated for 45 minutes prior to washing. FXIa (60 nM) was then addedto the antibody-coated beads and incubated for 1 hour. Five washes werecarried out prior to the addition of 1 000 picomoles of aptamer librarywhich had been diluted to 4 nM in AFB and heated to 90° C. for 5 minutesbefore being cooled on ice. The folded library was then added to theimmobilized FXIa and incubated for 1 hour with end-over-end rotation ona Barnstead Thermolyne Labquake. Unbound aptamers were removed by fivewashes in AFB. Bound aptamers were extracted from protein/antibody/beadassemblies using phenol: chloroform: isoamyl alcohol (71:24:1, vol/vol,saturated with Tris-Cl, Thermo Fisher Scientific) and precipitated with⅔ vol/vol absolute ethanol to conclude the selection round. The pool ofselected aptamers was then PCR-amplified using a high-fidelityheat-stable DNA polymerase (Phusion; Thermo Fisher Scientific) in anasymmetric PCR protocol in which 13-fold more primer A than primer B wasemployed; the ssDNA sense strand was then purified by preparativeagarose gel electrophoresis using a 2% (w/vol) agarose gel and anUltrafree-DA centrifugal filter unit (Millipore Sigma, Billerica, Mass.)to produce the amplified aptamer library and conclude the firstselection round (Round 1). for Round 3, selection stringency wasincreased by switching from AFB to stringent wash buffer (SWB; 20 mMTris-HCl pH 7.4, 4 M NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 0.005%Tween 20) and gradually reducing the incubation time of the library withFXIa such that it was only 15 min in Round 10.

After five rounds of positive selection, the resulting amplified librarywas combined with beads, biotinylated anti-FXIa, and FXIa as describedabove, and negatively selected using recombinant His-tagged KunitzProtease Inhibitor domain of human protease nexin 2 (KPI, 63 aminoacids) expressed in Pichia pastoris yeast and purified exactly aspreviously described (Navaneetham et al., 2005), at a concentration of6.8 μM, to block the FXIa active site. Washing and incubations were asdescribed above for positive selection, except that aptamers from theamplified library not binding to the KPI-FXIa-antibody-bead assemblieswere magnetically separated and combined with fresh FXIa, anti-FXIantibodies and magnetic beads to start the next round of selection.Rounds six through ten of SELEX thus combined positively selectingaptamer candidates with unblocked FXIa and negatively selecting againstaptamer candidates binding to KPI-blocked FXIa or anti-FXI antibodies ormagnetic beads.

High-Throughput Sequencing.

High-throughput (also known as deep) sequencing was employed tocharacterize the selected aptamer populations following four and tenrounds of SELEX. Single-stranded aptamers generated by asymmetric PCRwere PCR-amplified using forward primer 5′-AATGATACGGC GACCACCGAGATCTACACTA GATCGCACAC TCTTTCCCTA CACGACGCTC TTCCGATCTN NNNGAATTCTAATACGACTC ACTATA-3′ (SEQ ID NO: 42) and reverse primer 5′-CAAGCAGAAGACGGCATACG AGATTCGCCT TAGTGACTGG AGTTCAGACG TGTGCTCTTC CGATCTCGATGTGTTGGACA AGCAGAAGAC GGCATACGAG ATTCGCCTTA GTGACTGGAG TTCAGACGTGTGCTCTTCCG ATCTCGATGT GTTGGACGCC GC-3′ (SEQ ID NO: 43). The resultingamplicons were sequenced using an Illumina Miseq DNA sequencer at theFarncombe Metagenomics Facility, McMaster University. The raw sequencingdata was processed using Illumina's Basespace online NGS platform fortagged sequence pool sorting, and to ensure sequence data output wasconverted to FASTQ format. Further data processing was as described(Gysbers et al., 2015).

Aptamers.

The full length FELIAP aptamer sequence was determined to be5′-GAATTCTAAT ACGACTCACT ATAAACCTAT CGGACTATTG TTAGTGATTT TTATAGTGTGCGTCCAACAC ATCG-3′ (SEQ ID NO: 1). A control aptamer of the same lengthbut scrambled DNA sequence (SCRAPT) was synthesized for comparativepurposes, with sequence 5′-TTCTAATACG ACTCACTATA AGGGAGGGCA GTGGGATGGCGTTAGTGAGG GAGGGTGTGG GGCGTCCAAC ACAT-3′ (SEQ ID NO: 44). To generatetruncated versions of FELIAP, nucleotides from both the 3′ and 5′ endswere sequentially removed as depicted schematically in FIG. 6A andpresented in Table 4. Before use, all aptamer preparations were dilutedinto AF buffer, 10 mM Tris-Cl, 1 mM EDTA pH 8.0, Tris-buffered saline,or PPNE kinetics buffer (20 mM sodium phosphate, 100 mM NaCl, 0.1 mMEDTA, 0.1% polyethylene glycol (PEG) 8000, pH 7.4). The diluted aptamerswere refolded by heating to 90° C. for 5 min and then cooled for 10 minon ice.

Chromogenic Assay.

Assays were performed in a 96-well flat bottom microtiter plate (CorningIncorporated, Corning, N.Y.) at 37° C. in reaction buffer PPNE.Reactions (200 μL) contained 1 nM FXIa, 90 μM chromogenic substrateS2336 and aptamer concentrations ranging from 0.78 to 10 μM. The rate ofsubstrate hydrolysis was recorded at 405 nm on an ELx808 AbsorbanceMicroplate Reader (Biotek, Winooski, Vt., USA).

FXIa-Mediated FIX Activation.

FIX (6.2 μM) was incubated with 2 nM FXIa in the presence of 10 μMFELIAP, 10 μM scrambled DNA or 2.6 μM KPI in TBS supplemented with 5 mMCaCl₂ at 37° C. Samples were incubated for 30 min. Following the 30 minincubation, the reactions were quenched using Sodium Dodecyl Sulphate(SDS) polyacrylamide gel electrophoresis (SDS-PAGE) loading buffercontaining dithiothreitol and electrophoresed on a 12%SDS-polyacrylamide gel. Protein bands were visualized by staining withCoomassie Brilliant Blue.

Inhibition of FXIa by Antithrombin.

FXIa (200 nM) was pre-incubated with 10 μM FELIAP or SCRAPT at 37° C.for 5 minutes, and then combined with 2 μM purified human antithrombinin the presence of 2 U/mL sodium heparin (Sigma-Aldrich) for a further 1or 5 minutes. At the end of the antithrombin incubation time, sampleswere quenched and subjected to electrophoresis as described above.

FXI Activation Assay.

FXI (700 nM) in PPNE buffer was reacted with 70 nM thrombin in thepresence or absence of 1 mg/L dextran sulphate (molecular weight 500kDa) at 37° C. for 30 minutes in the presence or absence of 10 μM FELIAPor SCRAPT or 1.5 μM recombinant His-tagged hirudin variant 3 (HV3)(Sheffield et al., 2001). At the conclusion of the reaction samples werequenched and subjected to electrophoresis as described above.

Thrombin Generation Assay.

Thrombin generation assays (TGA) were performed using either humannormal pooled plasma (NPP) or FXI-depleted plasma (FXI-DP),(Haematologic Technologies, Essex Junction, Vt., USA) with or withoutsupplementation with FXIa. Three variations of TGA were performed,using: human normal pooled plasma (NPP) activated with silicates; FXI-DPactivated with tissue factor; and FXI-DP supplemented with FXIa. All TGAreactions were performed in black flat bottom 96-well microtiter plates(Greiner Bio One). In the first protocol, 40 μL of NPP (diluted 1:5 inphosphate-buffered saline (PBS) was mixed with 10 μL of 300 μM FELIAP orSCRAPT) or PBS, 10 μL of APTT reagent (PTT-A, Diagnostica Stago, silicaactivator diluted 1:20 in PBS), 71 μL of 2 mM TGA substrate solutioncontaining calcium chloride, and 15 μL of PBS, to comprise a totalreaction volume of 100 μL. In the second protocol, the APTT reagent wassubstituted with the prothrombin time reagent Innovin (recombinant humantissue factor (Dade Behring, Deerfield, Ill., USA) and NPP wassubstituted with FXI-DP. His-tagged recombinant hirudin variant 3purified from Pichia pastoris (Sheffield et al., 2001) served as apositive control for TGA inhibition. In the third protocol, 1 μM FELIAP,SCRAPT, or KPI were combined with 0.71 nM FXIa (10 μL) and pre-incubatedfor 5 min at room temperature prior to combination with FXI-DP andactivation with APTT reagent as in the first protocol. In all protocols,thrombin generation was then followed at 37° C. for 60 min at 1 minintervals using a Fluoroskan Ascent plate reader (Thermo Scientific) setto a wavelength of 380/460 nm. Data was imported into a Microsoft Excelevaluation spreadsheet (www.technoclone.com) for analysis and derivationof TGA test parameters.

Modified APTT assay. APTT assays were performed using a STart 4coagulometer (Diagnostica Stago) with some modifications. For thereactions, 5 μL of aptamer or PBS buffer control (FELIAP or SCRAPT, 30μM) was heat-denatured and combined with an equal volume of varyingconcentrations of purified human FXIa at 37° C. for 3 min. APTT reagent(APTT-XL, 50 uL) was separately preincubated with 45 μL FXI-deficienthuman plasma under the same conditions, and then clotting was initiatedby mixing both sets of components with 50 μL of 71 mM CaCl₂ and theclotting time was determined.

Surface Plasmon Resonance (SPR).

Kinetic measurements of aptamer binding were analyzed by SPR on aBiacore T200 (GE Healthcare). To immobilize the aptamer, a CM5 sensorchip (GE Healthcare) was activated with a 0.2 MN-ethyl-N′-(dimethylamino-propyl) carbodiimide (EDC) and 0.05 MN-hydroxysuccinimide (NHS) (Sigma) solution, followed by binding ofstreptavidin (0.2 mg/ml, pH 4.5). FELIAP and SCRAPT (3′ biotinylated) in100 mM HEPES (pH 7.4), 150 mM NaCl, 0.01% Tween 20 were immobilized onthe streptavidin-coated chips at a flow rate of 10 μL/min to 100response units (RU). Flow cells were regenerated with 0.2% SDS (w/vol).FXIa at concentrations varying from 15.671 nM to 500 nM was injected ata flow rate of 50 μl/min for 180 s to monitor association, and HEPESbuffer for 1800 s to monitor dissociation. The signal from theSCRAPT-immobilized flow cell was used as a reference and subtracted fromthe signal arising from FXIa binding to the FELIAP-immobilized cell.Binding of FXIa and aptamer was quantified by global analysis of on andoff rates using the Langmuir 1:1 binding model, as determined with theinstrument's software provided by the manufacturer (Biacore). Allexperiments were done in triplicate.

Selection of FXIa-Binding Aptamer from a Combinatorial Library.

The objective was to select FXIa-inhibiting aptamers from a largelibrary of ssDNA molecules 80 nucleotides in length containing aninternal randomized 40 nucleotide region flanked by primer bindingsites. Such a library theoretically contains 4⁴⁰ different DNAmolecules. An in vitro aptamer selection protocol was employed.Initially, only positive selection was employed to enrich for aptamersbinding to FXIa. After 4 and 10 rounds of selection, no inhibition ofFXIa-mediated amidolysis was noted when the selected aptamer pool wasintroduced into the reaction (data not shown). Accordingly, theselection protocol was modified by the addition of alternating positiveand negative selection steps and rescreened the initial library. Themodified protocol included negative selection of aptamers binding to anycomponent of the FXIa-antibody-bead assemblies except the FXIa activesite, by introducing the FXIa active site-binding, small proteininhibitor KPI (Navaneetham et al., 2005), after Round 4. In contrast tothe initial results, after Round 10, a small but reproducible reductionin amidolysis was observed in the presence of the selected aptamer pool.

Sequencing of the pool after Round 4 indicated that the number of uniquesequences had been reduced to 289; Table 3 shows the ten most abundantof these sequences. None elicited any inhibition of FXIa activity whentested individually. The majority of these abundant aptamer sequencescontained variable sequences of 21 to 23 nucleotides, rather than the 40nucleotide variable sequences present in the initial library. By Round10, the selected pool contained only 79 different sequences, as judgedby high-throughput sequencing; eight of the ten most abundant sequenceswere 36-40 nucleotides long. Two abundant sequences, Apt10-1 andApt10-3, were also found in the Round 4 pool (Apt4-1 and Apt4-2). Whenthe ten most abundant sequences in the Round 10 pool were testedindividually, a single aptamer, Apt10-10, was found to inhibitFXIa-mediated cleavage of chromogenic substrate S2366. This tenth mostabundant aptamer sequence in Round 10 was designated Factor ELeven(a)Inhibitory Aptamer (FELIAP). When the initial screen of the aptamerlibrary that involved only positive selection with FXIa was continued inparallel to the positive/negative approach, both anti-FXIa activity ofthe pool and the presence of FELIAP in the pool were noted at Round 20(data not shown).

TABLE 3 DNA sequence of the core of the aptamers (name and SEQ ID NOs,column 3) isolated after 4 or 10 rounds of screening (column 1)corresponding to the N₄₀ variable domain in the original aptamer library(5′-GAATTCTAAT ACGACTCACT ATA-N₄₀- GCGTCCAACA CATCG-3′ or SEQ ID NO: 29)is given in column 2. Whether (+) or not (−) the aptamer had inhibitoryactivity when introduced into FXIa-mediated amidolysis of S2366 is shownin column 4. The ten most abundant sequences obtained after Rounds 4 or10 are shown in rank order; NT signifies not tested. Name, (SEQ FXIaRound N₄₀ Variable sequence ID NO) Inhibition 4 GCGTCCAACACATCGTATTCATApt4-1 (45) − 4 TGGGATGGCGTGGGAGGGCTGTAGGGAGCGTTC Apt4-2 (46) NT AGTGGGT4 GGGAGGGCGTGGATGGCTGGTGTGAGGTCTTGT Apt4-3 (47) − GTTTGTT 4GGGAGCGTTCAGTGGGT Apt4-4 (48) NT 4 GCGTCCAACACATCGGATGATAT Apt4-5 (49)NT 4 TGCGTCCAACACATCGTATTCAT Apt4-6 (50) NT 4TGGGATGGCGTGGGAGGGCTGTAGTGAGCGTTC Apt4-7 (51) NT AGTGGGT 4CGTCCAACACATCGTATTCAT Apt4-8 (52) NT 4 CTTGCCCACTATCGACTTCACC Apt4-9(53) NT 4 GCGTCCAACACATCGTAAGTA Apt4-10 (54) NT 10GCGTCCAACACATCGTATTCAT Apt10-1 (45) − 10 CACTGCGTCCAACACATCGTATTCATApt10-2 (55) − 10 TGGGATGGCGTGGGAGGGCTGTAGGGAGCGTTC Apt10-3 (46) −AGTGGGT 10 TGGGATGGCGTGGGAGGGCTGTAGTGAGCGTTC Apt10-4 (51) − AGTGGGT 10GGGAGGGCGTGGATGGCTGTTGTGAGGTCTTGT Apt10-5 (56) − GTTTGTT 10TGGGATGGCGTGGGAGGGCTGTAGGGAGCGTTT Apt10-6 (57) − AGTGGGT 10TGGGATGGCGTGGGAGGGCTGTAGTGAGCGTTC Apt10-7 (58) − ATTGGGT 10GGGAGGGCGTGGATGGCTGGTGTGAGGTCTTGT Apt10-8 (47) − GTTTGTT 10TGGGATGGCGTGGGAGGGCTGTAGTGAGCGTTT Apt10-9 (59) − AGTGGGT 10AACCTATCGGACTATTGTTAGTGATTTTTATAGTGT Apt10-10 (24) +

Comparison of FELIAP to related aptamer candidates. Comparison of FELIAPto the other 78 sequences of lesser abundance found in Round 10 revealedfive related aptamers, four of which (Apt10_A through D) were closelyrelated, differing in at most two of 36 positions; the fifth (Apt10_E)was only distantly related by virtue of T-rich areas (38.9%identical—see Table 3). The aligned sequences are shown in Table 1. Whenthese aptamers were tested for their ability to inhibit FXIa amidolysis,FELIAP was found to be the most active and Apt10_E the least active,although all six selected aptamers showed greater inhibitory activitythan SCRAPT (FIG. 1A). Modeling of predicted secondary structure usingthe Mfold web server showed that the portion of FELIAP corresponding tothe variable part of the aptamer library likely adopted an extendedstem-loop structure containing a bulge separated by two small loops(FIG. 1B). Three of four substitutions correlating with reducedanti-FXIa activity were found in a predicted extended 9 base pair stemstructure at the distal end of this structure, while the substitution inAPT10_C was at the very apex of the predicted hairpin. As FELIAP was themost potent inhibitor identified in the library, it was employed in allsubsequent experiments.

Kinetic Characterization of FELIAP as an Inhibitor of FXIa-MediatedAmidolysis and Clotting.

The mode of inhibition of FELIAP was investigated by fixing theconcentrations of chromogenic substrate and FXIa and increasing theconcentration of FELIAP and measuring the rate of substrate cleavage. Adose-dependent reduction in the reaction rate was observed as FELIAP wasincreased from 0 to 20 μM, while the same concentrations of SCRAPT hadno inhibitory effect (FIG. 2A). Next, the concentration of chromogenicsubstrate S2366 was varied while keeping FXIa constant at differentconcentrations of FELIAP (FIG. 2B). The reaction demonstratedcompetitive inhibition, as suggested by the apparent lack of alterationof the maximum reaction velocity (FIG. 2B) with increasing FELIAPconcentrations, and by the common y intercept on the Lineweaver-Burketransformation of the velocity versus substrate curves for increasingFELIAP concentrations (FIG. 2C). Fitting the curves to a competitiveinhibition model yielded an estimated Ki for FELIAP of 29 μM bynon-linear regression.

Prior to examining the inhibitory effects of FELIAP on FXIa-mediatedinteractions with specific macromolecular substrates, the capacity ofFELIAP to inhibit FXIa-induced clotting in plasma was examined. FXIa waspre-incubated with buffer, FELIAP, or SCRAPT, and combined withFXI-deficient plasma and APTT reagent containing kaolin and cephalin,and recalcified. As shown in FIG. 2D, at all three FXIa concentrationstested, FELIAP delayed plasma clot formation to a greater extent thanSCRAPT or buffer controls.

Effects of FELIAP on Macromolecular Reactions of FXI and FXIa.

FXIa activates FIX by cleaving two peptide bonds, one at Arg145-Ala146,and the other at Arg180-Val181 of the FIX polypeptide30. This reactionliberates a glycosylated activation peptide (Ala146-Arg180, 10 kDa), anda disulphide-linked γ-carboxylated light chain (Tyr1-Arg145, 71 kDa) andheavy chain (Val181-Thr405, 30 kDa)31,32. An intermediate productcomprised of the activation peptide linked to the heavy chain(Ala146-Thr405, 40 kDa) may also be detected. FIG. 3A shows thatFXIa-mediated FIX activation was unaffected by SCRAPT (compare heavy andlight chains, lanes 1 and 2), while introduction of excess FELIAP (lane3) or KPI (lane 4) reduced FIXa generation to background levels (lane 5,no FXIa added).

FXIa also reacts with its natural inhibitor, antithrombin, in a reactionthat is accelerated by heparin, to form a denaturation-resistant complexbetween the FXIa active site and the reactive centre of antithrombin, inwhich the light chain of FXIa (Ile370-Val607) is joined to antithrombinresidues His1-Arg393 via an acyl linkage10,33. FIG. 3B shows SDS-PAGEevidence of this 90 kDa complex after 1 or 5 minutes of reaction (lanes2 and 3) and in the presence of excess SCRAPT (lanes 6 and 7) but not inthe presence of excess FELIAP (lanes 4 and 5).

Having demonstrated that FELIAP inhibited the action of FXIa on twomacromolecular substrates, FIX and antithrombin, it was then determinedif it had any effect on the activation of FXI by thrombin. In thepresence of cofactor dextran sulphate (FIG. 3C, lane 3), but not itsabsence (lane 2), FXI was efficiently activated into FXIa, as previouslyreported. No inhibition of this reaction was noted in the presence ofeither excess FELIAP (lane 4) or SCRAPT (lane 5), but activation of FXIwas abrogated in the presence of excess hirudin, a potent thrombininhibitor (lane 6). FELIAP therefore inhibited FXIa-dependent but notFXI-dependent reactions.

Inhibition of Thrombin Generation in Plasma by FELIAP.

Recalcified dilute normal human pooled plasma, in which the contactpathway of coagulation was activated by micronized silica, was employedto assess the effects of FELIAP on thrombin generation. The thrombingeneration assay (TGA) provides information on the timing and kineticsof thrombin generation in plasma using a thrombin-specific fluorogenicsubstrate and calibrators unaffected by fibrin clot formation, usingstandardized analytic methods and parameters35. As shown in FIG. 4A,FELIAP had greater effects on thrombin generation in recalcified diluteplasma than SCRAPT. Firstly, 30 μM FELIAP prolonged the lag time ofthrombin generation relative to either 30 μM SCRAPT or buffer controls(data not shown). Similarly, FELIAP reduced the endogenous thrombinpotential (ETP; the area under the thrombin generation curve) by3.2-fold, versus 1.4-fold for SCRAPT (FIG. 4B; both reductions p<0.001).Finally, the mean time to peak thrombin was increased 1.5-fold by FELIAPbut was unaffected by SCRAPT (FIG. 4C).

The partial anticoagulant activity observed for SCRAPT insilica-activated TGA using normal pooled plasma was also noted for othersingle-stranded oligonucleotides of similar length (70-80 nucleotides;data not shown) at equimolar concentrations. To ascertain whether or notthis phenomenon was related to FXIa inhibition, TGA was repeated,substituting FXI-depleted plasma for normal plasma and tissue factor forsilicates as activators (FIG. 4D). SCRAPT or FELIAP exhibitedindistinguishable 1.24- to 1.27-fold reductions in endogenous thrombinpotential relative to buffer under these conditions (FIG. 4E), butunaltered times to peak thrombin (FIG. 4F). In contrast, the specificthrombin inhibitor hirudin, at 200 nM, elicited a significantly greaterreduction in ETP than 30 μM FELIAP or SCRAPT, and extended the time topeak thrombin by 3.1-fold (p<0.001 versus FELIAP, SCRAPT or buffer, FIG.4F). SCRAPT or FELIAP effects were eliminated when the aptamers (1.0 μM)were combined with 0.71 nM FXIa and then combined with FXI-depletedplasma activated by silicates (FIGS. 4G-1). In contrast, substitution ofKPI for either aptamer significantly reduced the ETP and the time topeak thrombin (FIGS. 4G-1). FELIAP therefore inhibited TGA to a greaterextent than SCRAPT in plasma activated via the contact pathway, but notthe extrinsic pathway, but to a considerably lesser extent than KPI.

Use of Surface Plasmon Resonance (SPR) to Characterize FEL/AP Binding toFXIa.

FELIAP and SCRAPT were biotinylated at their 3′ ends and immobilized ona streptavidin-coated gold chip. Immobilized SCRAPT served as thereference cell. When increasing concentrations of FXIa from 0 to 500 nMwere flowed over these surfaces, increasing response unit bindingisotherms were generated when the difference between immobilized FELIAPand immobilized SCRAPT binding was plotted (FIG. 5). It should be notedthat the maximum response for SCRAPT binding did not exceed 71 responseunits at 500 nM FXIa, 1800 seconds (data not shown). The net bindingisotherm was characterized by relatively rapid association kinetics andvery slow dissociation kinetics. Analysis of these isotherms yieldedvalues for dissociation rate constant (Kd) and the association rateconstant (Ka), permitting calculation of the equilibrium bindingconstant KD, which is Kd divided by Ka. Ka values of 5.2±0.1×104 M-1S-1, Kd values of 9.5±0.1×10-5 S-1, and KD values of 1.8±0.1×10-9 M wereobtained (mean±SD of three determinations).

Effects of Progressive Truncation of FELIAP on Inhibition ofFXIa-Mediated Amidolysis.

To determine the minimum active sequence of FELIAP required forinhibition of FXIa, several truncated forms of FELIAP were synthesized,using the predicted structure of the aptamer to delete predicted loopsand stems progressively. The truncated sequences ranged from 32 to 64nucleotides long (compared to 74 for full-length FELIAP), weredesignated FELIAP_X (where X=32, 38, 49, 56 and 64), and constituted 5′and 3′ deletion mutants of FELIAP (see Table 4 as well as FIG. 6A). Asshown in FIG. 6B, deletion of two predicted loop structures and anintervening short stem (in FELIAP_38) had no effect on anti-FXIaactivity. However, reduction of the predicted terminal stem structurefrom seven (in FELIAP_38) to four base pairs (in FELIAP_32) reducedanti-FXIa activity to background levels equivalent to SCRAPT.

TABLE 4 DNA sequence of truncated aptamers used to provide the resultsof FIG. 6. The underlined regions of the sequences refer to the“constant primer binding” sites (e.g., the 5′ wing or the 3′ wing) whilethe italicized regions refer to the core region. Name SEQ (SEQ ID ID NO)Nucleotide sequence NO FELIAP GAATTCTAATACGACTCACTATA AACCTATCGGACTA 1TTGTTAGTGATTTTTATAGTGT GCGTCCAACACATCG FELIAP_64 CTAATACGACTCACTATAAACCTATGGA 60 CTATTGTTAGTGATTTTTATAGTGT GCGTCCAACAC FELIAP_55TACGACTCACTATA AACCTATCGGACTA 61 TTGTTAGTGATTTTTATAGTGT GCGTC FELIAP_49GACTCACTATA AACCTATCGGACTATTG 62 TTAGTGATTTTTATAGTGT GC FELIAP_42CACTATA AACCTATCGGACTATTGTTAG 63 TGATTTTTATAGTG FELIAP_38 CTATAAACCTATCGGACTATTGTTAGTG 64 ATTTTTATAG FELIAP_32 TAAACCTATCGGACTATTGTTAGTGATT 65 TTTA

Aptamers from combinatorial libraries can be selected to bind virtuallyany protein of interest. Here, the selection and characterization ofFELIAP, a FXIa-binding DNA aptamer was described. The protocol usedfavoured the selection of aptamers binding at or near the active site ofFXIa over those binding to other portions of the enzyme. Several linesof indirect evidence support the conclusion that this strategy wassuccessful. Firstly, FELIAP acted as a competitive inhibitor ofchromogenic substrate S2366, which is a tripeptide nitroanilide compound(pyroglutamyl-prolyl-arginyl-p-nitroanilide) which must, by virtue ofits small size, enter the interior of the FXIa active site pocket to becleaved and liberate the coloured product nitroanilide. Secondly, itinhibited two different FXIa-dependent reactions: FXIa-mediatedactivation of FIX; and FXIa-mediated formation of FXIa-antithrombincomplexes. While this observation does not per se exclude an allostericeffect of FELIAP on FXIa, it renders it less likely than active sitebinding. Thirdly, FELIAP had no effect on FXI activation, furtherindicating specificity for FXIa, one of whose cardinal features is theactive site. Taken together, these data suggest that FELIAP bindsspecifically to FXIa at or near its active site, with high affinityconsistent with the observed nanomolar KD.

KPI was employed to obscure the active site of FXIa inKPI-FXIa-anti-FXI-bead assemblies for negative selection, reasoning thatdepletion of the aptamer library of candidates binding to any of theconstituent parts of these assemblies would enrich for those binding theFXIa active site. KPI is the Kunitz protease inhibitor domain ofprotease nexin 2, which is an isoform of the β-amyloid precursor protein(APP) secreted from α-granules on platelet activation. The 57 amino acidKPI domain accounts for all of the FXIa-inhibitory activity of APP36 andhas been crystallized in complex with FXIa28. The crystal structurerevealed that two loops in KPI formed extensive contacts with FXIa; inparticular residues in the KPI Thr11-Arg20 loop extend into thesubstrate pocket, rationalizing the high affinity binding evidenced byreported Ki values of 300 500 μM. While it may seem somewhat circular touse a polypeptide active site inhibitor to identify a ssDNA aptamerinhibitor, aptamers provide multiple potential advantages overproteinaceous inhibitors with respect to immunogenicity, cost ofproduction, antidote generation, and the potential to fine-tune bindingvia mutagenesis.

While to our knowledge no aptamers to FXI or FXIa have been previouslydescribed in the biomedical literature, several aptamers to othercoagulation factor targets have been isolated.

Among RNA aptamers, RNA 16.3 bound FVII or FVIIa with KD values of 10-13nM, inhibiting the enzyme by disrupting tissue factor-FVII(a) complexassembly. A truncated form of a selected RNA aptamer designated 9.3t wasfound to bind FIXa with a KD of 0.58 nM38, specifically at an extendedsubstrate binding position, or exosite. RNA11F7t aptamer bound FXa witha reported KD of 1.1 nM and inhibited prothrombinase activity byinterfering with the interaction between FXa and FVa, while R4CxII-Itbound FXII or FXIIa with KD values of 8.9 and 0.4 nM, respectively, byinterfering with FXII and anionic binding. RNAR9D-14T bound thrombin orprothrombin with KD values of 1 or 10 nM via anion binding exosite I.With respect to DNA aptamers, the only ones described to date thattarget coagulation factors are HD1 and HD22, which bind prothrombin withKD values of 7.1 and 2.4 nM, via exosites I and II, respectively. Allpreviously described aptamers targeting coagulation factors, therefore,act at sites distinct from the active site of these serine proteases.The fact that FELIAP, which acts at or near the active site of FXIa, mayhave been facilitated by the use of KPI in negative selection, asscreening protocols employed in previously published studies used onlypositive selection. Although the time- and labour-intensive nature ofaptamer library screening precluded a systematic analysis, anecdotallyFELIAP selection was observed within ten rounds of screening withpositive and negative steps, but not until twenty rounds of screeningwith positive steps alone.

Although SELEX and SELEX-related aptamer screening protocols have beenemployed to isolate aptamers that bind to their targets with highaffinity, numerous limitations need to be overcome for successfulapplication of this biotechnological approach. These includeinterference from non-degenerate sequences flanking the variable regionsin the aptamer library; non-specific retention of sequences not bindingthe desired target; and accumulation of amplification artifacts andartifactual sequences arising from library regeneration. Many examplesof such artifacts were observed using high throughput sequencing afterRound 4, some of which persisted to Round 10, as well as the selectionof aptamers with barely detectable affinity for FXIa (e.g. Apt10_E). Ourresults are therefore consistent with the known limitations of SELEX.

Inspection of the Mfold generated secondary structural foldingprediction showed that the extended stem-loop structure predicted forFELIAP encompassed more than the variable region nucleotides 24 to 59.FELIAP_38 retained all of full-length FELIAP's anti-FXIa activity, butonly three additional base pairs in a predicted terminal stem thannon-inhibitory FELIAP_32. These results, taken together with the findingthat mutation of the guanosine residue at the predicted turn of thehairpin at nucleotide 40, support the secondary structure model andlikely indicate that the AT-rich stem disrupted in FELIAP_32 must bestabilized by additional A-T hydrogen bonds or by the C-G bondingbetween residues 19 and 56 in FELIAP_38. It is likely that this stemstabilizes the “loop-bulge-loop” conformation of FELIAP_38 for directcontact with FXIa. Localization of the aptamer within the FXIa activesite by cross-linking, modeling or co-crystallization will in future beused to test this working hypothesis and to provide clues to how toincrease its potency as an inhibitor to match its high affinity as aFXIa ligand.

While the invention has been described in connection with specificembodiments thereof, it will be understood that the scope of the claimsshould not be limited by the preferred embodiments set forth in theexamples, but should be given the broadest interpretation consistentwith the description as a whole.

REFERENCES

-   Buller H R, Bethune C, Bhanot S, Gailani D, Monia B P, Raskob G E,    Segers A, Verhamme P, Weitz J I; FXI-ASO TKA Investigators. Factor    XI antisense oligonucleotide for prevention of venous thrombosis. N    Engl J Med. 2015 Jan. 15; 372(3):232-40.-   Gysbers, R., Tram, K., Gu, J. & Li, Y. Evolution of an Enzyme from a    Noncatalytic Nucleic Acid Sequence. Sci Rep 5, 11405 (2015).-   Navaneetham, D. et al. Structural and mutational analyses of the    molecular interactions between the catalytic domain of factor XIa    and the Kunitz protease inhibitor domain of protease nexin 2. J Biol    Chem 280, 36165-36175 (2005).-   Sheffield, W. P., Smith, I. J., Syed, S. & Bhakta, V. Prolonged in    vivo anticoagulant activity of a hirudin-albumin fusion protein    secreted from Pichia pastoris. Blood Coagul Fibrinolysis 12, 433-443    (2001).-   Tripodi A. Thrombin generation assay and Its application in the    clinical laboratory. Clin Chem. 2016 May; 62(5):699-707.-   Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential    enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science    249, 505-510 (1990).

What is claimed is:
 1. An aptamer having the structure of formula (I):5′-5W—C-3W-3′  (I) wherein: “-” refers to a nucleotide bond; 5W has thefollowing first nucleic acid sequence: (SEQ ID NO: 66)N₁₇N₁₈N₁₉N₂₀N₂₁N₂₂N₂₃N₂₄N₇₁N₂₆N₂₇N₂₈AN₂₉TN₃₀TA;

C has the following second nucleic acid sequence: (SEQ ID NO: 68)5′-AACCTATN₁N₂N₃ACTATTN₄TN₅AN₆TN₇ATTTTTAN₈AN₉-3′;

3W can be present or absent and when present has the following thirdnucleic acid sequence: (SEQ ID NO: 67)5′-N₃₁N₃₂N₃₃N₃₄N₃₅N₃₆N₃₇N₃₈N₃₉N₄₀N₄₁-3′;

the nucleotides at positions 14 to 18 of SEQ ID NO: 66 are present; thenucleotides at positions 1 to 33 of SEQ ID NO: 68 are present; thenucleotides at position 1 to 2 (AA) of SEQ ID NO: 68 are capable of basepairing with the nucleotides at position 28 to 27 (TT) of SEQ ID NO: 68;the nucleotides at position 6 to 8 (ATC) of SEQ ID NO: 68 are capable ofbase pairing with the nucleotides at position 25 to 23 (TAG) of SEQ IDNO: 68; the nucleotides at position 11 to 14 (ACTA) of SEQ ID NO: 68 arecapable of base pairing with the nucleotides at position 22 to 19(TN₆AN₅) of SEQ ID NO: 68; and the nucleotides at position 14 to 18(N₂₉TN₃₀TA) of SEQ ID NO: 66 are capable of base pairing with thenucleotides at position 33 to 29 (N₉AN₈AT) of SEQ ID NO:
 68. 2. Theaptamer of claim 1 having the nucleic acid sequence of SEQ ID NO:
 64. 3.The aptamer of claim 1, wherein, the nucleotides at positions 12 and 13of SEQ ID NO: 66 and at position 34 and 35 of SEQ ID NO: 68 are present,and the nucleotides at position 12 and 13 (N₂₈A) of SEQ ID NO: 66 arecapable of base pairing with the nucleotides at position 35 to 34 (N₁₀T)of SEQ ID NO:
 68. 4. The aptamer of claim 3 having the nucleic acidsequence of SEQ ID NO:
 63. 5. The aptamer of claim 2, wherein thenucleotides at position 8 to 11 of SEQ ID NO: 66, at position 1 to 5 ofSEQ ID NO: 67 and at position 36 of SEQ ID NO: 68 are present, and thenucleotide at position (N₂₄) of SEQ ID NO: 66 is capable of base pairingwith the nucleotide a position 2 (N₃₂) of SEQ ID NO:
 67. 6. The aptamerof claim 5 having the nucleic acid sequence of SEQ ID NO:
 62. 7. Theaptamer of claim 3, wherein the nucleotides at position 5 to 7 of SEQ IDNO: 66 and at position 3 to 5 of SEQ ID NO: 67 are present, and thenucleotides at position 6 to 7 (N₂₂N₂₃) of SEQ ID NO: 66 are capable ofbase pairing with the nucleotides at positions 4 to 3 (N₃₄N₃₃) of SEQ IDNO:
 67. 8. The aptamer of claim 7 having the nucleic acid sequence ofSEQ ID NO:
 61. 9. The aptamer of claim 2, wherein nucleotides 1 to 4 ofSEQ ID NO: 66 and nucleotides 6 to 11 of SEQ ID NO: 67 are present. 10.The aptamer of claim 9 having the nucleic acid sequence of SEQ ID NO:60.
 11. The aptamer of claim 1, wherein 5W has the following firstnucleic acid sequence: (SEQ ID NO: 25)5′-N₁₂N₁₃N₁₄N₁₅N₁₆N₁₇N₁₈N₁₉N₂₀N₂₁N₂₂N₂₃N₂₄N₇₁N₂₆ N₂₇N₂₈AN₂₉TN₃₀TA 3′;

C has the following second nucleotide sequence: (SEQ ID NO: 23)5′-AACCTATN₁N₂N₃ACTATTN₄TN₅AN₆TN₇ATTTTTAN₈AN₉TN₁₀ N₁₁-3′.

3W is present and has the following third nucleotide sequence: (SEQ IDNO: 27) 5′-N₃₁N₃₂N₃₃N₃₄N₃₅N₃₆N₃₇N₃₈N₃₉N₄₀N₄₁N₄₂N₄₃N₄₄N₄₅₋ 3′;

“-” refers to a nucleotide bond; the nucleotides at position 1 to 36 ofSEQ ID NO: 23, at position 6 to 23 of SEQ ID NO: 25 and at position 1 to11 of SEQ ID NO: 27 are present; the nucleotides at position 11 to 13(ACTA) of SEQ ID NO: 23 are capable of base pairing with the nucleotidesat position 22 to 19 (TN₆AN₅) of SEQ ID NO: 23; the nucleotides atposition 6 to 8 (ATN₁) of SEQ ID NO: 23 are capable of base pairing withthe nucleotides at position 25 to 23 (TAN₇) of SEQ ID NO: 23; thenucleotides at position 1 to 2 (AA) of SEQ ID NO: 23 are capable of basepairing with the nucleotides at positions 28 to 27 (TT) of SEQ ID NO:23; the nucleotides at position 17 to 23 (N₂₈AN₂₉TN₃₀T) of SEQ ID NO: 25are capable of base pairing with the nucleotides at positions 35 to 27of SEQ ID NO: 23 (N₁₀TN₉AN₈A); and the nucleotides at position 6 to 8(N₂₂N₂₃N₂₄) of SEQ ID NO: 25 are capable of base pairing with thenucleotides at position 4 to 2 (N₃₄N₃₃N₃₂) of SEQ ID NO:
 27. 12. Theaptamer of claim 11, wherein 5W has the nucleotide sequence of SEQ IDNO: 26, 3W has the nucleotide sequence of SEQ ID NO: 28 and/or C has thenucleotide sequence of SEQ ID NO:
 24. 13. The aptamer of claim 1 havingthe nucleotide sequence of SEQ ID NO: 1, SEQ ID SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:
 13. 14. Amethod for detecting Factor XIa in a sample, the method comprising: (i)contacting the aptamer of claim 1 with the sample; (ii) determining thepresence or the absence of a complex between Factor XIa and the aptamer;and (iii) detecting Factor XIa in the sample if the complex of step (ii)is determined to be present.
 15. The method of claim 14, furthercomprising, when the complex of step (ii) is determined to be present,(iv) quantifying the amount of Factor XIa in the sample based on theamount of the complex.
 16. A method of imaging a clot in a subject, themethod comprising: (i) administering the aptamer of claim 1 to thesubject; (ii) determining the presence or the absence of a complexbetween Factor XIa and the aptamer; and (iii) imaging a clot in thesubject at the location of the complex.
 17. A method of purifying FactorXIa from a sample, the method comprising: (i) contacting the aptamer ofclaim 1 with the sample; (ii) allowing for a complex between Factor XIaand the aptamer to form; and (iii) removing the complex from the sample.18. A method of determining the usefulness of a test agent for theprevention, treatment or the alleviation of symptoms of thrombosis in asubject, the method comprising: (i) contacting the test agent withFactor XIa to obtain a test level of the biological activity of FactorXIa, (ii) comparing the test level to a control level of the biologicalactivity of Factor XIa, the control level being derived from or obtainedby contacting Factor XIa with the aptamer of claim 1; and (iii)determining the test agent as being useful for the prevention, treatmentor the alleviation of symptoms of thrombosis if the test level is equalto or lower than the control level.
 19. The method of claim 18, whereinstep (i) and/or (ii) comprises determining the biological activity ofFactor XIa by measuring the proteolytic activity of Factor XIa.
 20. Themethod of claim 18, wherein step (ii) comprises providing the aptamerand determining the control level of the biological activity of FactorXIa.